Buffer capacities of leaves, leaf cells, and leaf cell organelles in relation to fluxes of potentially acidic gases

Since environmental pollution by potentially acidic gases such as SO₂ causes proton release inside leaf tissues, homogenates of needles of spruce (Picea abies) and fir (Abies alba) and of leaves of spinach (Spinacia oleracea) and barley (Hordeum vulgare) were titrated and buffer capacities were determined as a function of pH. Titration curves of barley leaves were compared with titration curves of barley mesophyll protoplasts. From the protoplasts, chloroplasts and vacuoles were isolated and subjected to titration experiments. From the titration curves, the intracellular distribution of buffering capacities could be deduced. Buffering was strongly pH-dependent. It was high at the extremes of pH but still significant close to neutrality. Owing to its large size, the vacuole was mainly responsible for cellular buffering. However, on a unit volume basis, the cytoplasm was much more strongly buffered than the vacuole. Potentially acidic gases are trapped in the anionic form. They release protons when trapped. The magnitude of diffusion gradients from the atmosphere into the cells, which determines flux, depends on intracellular pH. In the light, the chloroplast stroma, as the most alkaline leaf compartment, has the highest trapping potential. Acidification of the chloroplast stroma inhibits photosynthesis. The trapping potential of the chloroplast is followed by that of the cytosol. Compared with the cytoplasm, the vacuole possesses little trapping potential in spite of its large size. It is particularly small in the acidic vacuoles of conifer needles. In the physiological pH range (slightly above neutrality), chloroplast buffering was about 1 microequivalents H⁺ per milligram chlorophyll per pH unit or 35 microequivalents H⁺ per milliliter per pH unit in barley or spinach chloroplasts. This compares with SO₂-generated H⁺ production of somewhat more than 1 microequivalent H⁺ per milligram chlorophyll per hour, which results from observed SO₂ uptake of leaves when stomata were open and the atmospheric SO₂ concentration was 0.4 microliters per liter (GE Taylor Jr, DT Tingey 1983 Plant Physiol 72: 237-244). At lower SO₂ concentrations, similar H⁺ generation inside the cells requires correspondingly longer exposure times.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Control of Intracellular pH in Chara corallina during Uptake of Weak Acid

Butyric acid was used to acidify the cytoplasm of cells of Characorallina in order to study the mechanisms that regulate intracellularpH. Butyric acid was found to enter the cell rapidly, predominantlyas the undissociated acid, and to dissociate in the cytoplasmto yield high concentrations of the butyrate anion. A rapidreduction in cytoplasmic pH was followed by partial recovery.The reductions in cytoplasmic pH resulting from butyrate accumulationwere small compared to the proton load calculated from the cytoplasmicbuffering capacity and intracellular dissociation of butyricacid. The cytoplasmic and vacuolar buffering capacities, calculatedfrom titration of cell extracts, were 17.9 and 0.5 mol m^–3per pH unit respectively. It was concluded that pH control in Chara during weak acid accumulationwas mainly due to membrane transport (active efflux) of protons.The factors which might determine the rate and extent of protonefflux, such as the energy supply and the availability of ionsfor charge balance, were examined. Butyrate strongly inhibitedphotosynthesis and caused a slight reduction in the rate ofrespiration. The mechanism of inhibition of photosynthesis isdiscussed in relation to the reported effects of weak acidson isolated chloroplasts. Key words: Cytoplasmic pH, weak acids, Chara     

Counting peptide-water hydrogen bonds in unfolded proteins

It is often assumed that the peptide backbone forms a substantial number of additional hydrogen bonds when a protein unfolds. We challenge that assumption in this article. Early surveys of hydrogen bonding in proteins of known structure typically found that most, but not all, backbone polar groups are satisfied, either by intramolecular partners or by water. When the protein is folded, these groups form approximately two hydrogen bonds per peptide unit, one donor or acceptor for each carbonyl oxygen or amide hydrogen, respectively. But when unfolded, the backbone chain is often believed to form three hydrogen bonds per peptide unit, one partner for each oxygen lone pair or amide hydrogen. This assumption is based on the properties of small model compounds, like N-methylacetamide, or simply accepted as self-evident fact. If valid, a chain of N residues would have approximately 2N backbone hydrogen bonds when folded but 3N backbone hydrogen bonds when unfolded, a sufficient difference to overshadow any uncertainties involved in calculating these per-residue averages. Here, we use exhaustive conformational sampling to monitor the number of H-bonds in a statistically adequate population of blocked polyalanyl-six-mers as the solvent quality ranges from good to poor. Solvent quality is represented by a scalar parameter used to Boltzmann-weight the population energy. Recent experimental studies show that a repeating (Gly-Ser) polypeptide undergoes a denaturant-induced expansion accompanied by breaking intramolecular peptide H-bonds. Results from our simulations augment this experimental finding by showing that the number of H-bonds is approximately conserved during such expansion⇋compaction transitions.     

Proton stoichiometric imbalance during algae photosynthetic growth on various nitrogen sources: toward metabolic pH control

The use of algae as a potential platform for fuels or biochemical production requires process design and control that can be implemented at agronomic scales. Toward achieving pH control in large unmixed systems, we present a rigorous set of direct measurements of non-buffered proton uptake and efflux during growth on ammonium and nitrate, observing nearly unit molar proton imbalance H⁺/OH^? respectively for these nitrogen sources. This proton imbalance can be shown to be consistent with the initial assimilation steps of nitrogen from glutamate to peptide bonds which indicates that the remainder of metabolism is largely net proton balanced. These results are refined by demonstrating pH balance for growth with incrementally fed nitric acid and ammonium hydroxide. In contrast to the typical assumption of simple charge balance, each displays a slight proton uptake (around 10 % excess) that is considerably lower than urea, which displayed a molar H⁺ uptake per N assimilated of up to 33 %. This work illustrates details of proton imbalance that have been largely obscured in laboratory work due to use of elevated CO₂ and its associated carbonate equilibrium. Combined with the recent demonstration of preferential, mutually exclusive assimilation of ammonium over nitrate in Chlorella and Chlamydomonas, these results provide the stoichiometry and dynamics of photosynthetic algae growth needed to implement large-scale pH control in the absence of buffering.     

Close agreement between deterministic versus stochastic modeling of first-passage time to vesicle fusion

Ca²⁺-dependent cell processes, such as neurotransmitter or endocrine vesicle fusion, are inherently stochastic due to large fluctuations in Ca²⁺ channel gating, Ca²⁺ diffusion, and Ca²⁺ binding to buffers and target sensors. However, previous studies revealed closer-than-expected agreement between deterministic and stochastic simulations of Ca²⁺ diffusion, buffering, and sensing if Ca²⁺ channel gating is not Ca²⁺ dependent. To understand this result more fully, we present a comparative study complementing previous work, focusing on Ca²⁺ dynamics downstream of Ca²⁺ channel gating. Specifically, we compare deterministic (mean-field/mass-action) and stochastic simulations of vesicle exocytosis latency, quantified by the probability density of the first-passage time (FPT) to the Ca²⁺-bound state of a vesicle fusion sensor, following a brief Ca²⁺ current pulse. We show that under physiological constraints, the discrepancy between FPT densities obtained using the two approaches remains small even if as few as ～50 Ca²⁺ ions enter per single channel-vesicle release unit. Using a reduced two-compartment model for ease of analysis, we illustrate how this close agreement arises from the smallness of correlations between fluctuations of the reactant molecule numbers, despite the large magnitude of fluctuation amplitudes. This holds if all relevant reactions are heteroreaction between molecules of different species, as is the case for bimolecular Ca²⁺ binding to buffers and downstream sensor targets. In this case, diffusion and buffering effectively decorrelate the state of the Ca²⁺ sensor from local Ca²⁺ fluctuations. Thus, fluctuations in the Ca²⁺ sensor’s state underlying the FPT distribution are only weakly affected by the fluctuations in the local Ca²⁺ concentration around its average, deterministically computable value.     

Simultaneous determination of ΔG, ΔH and ΔS by an automatic microcalorimetric titration technique. Application to protein ligand binding

A methodological study has been made with a syringe titration unit attached to an LKB batch microcalorimeter. The presicion and accuracy of the instrument assembly have been evaluated by neutralization reactions and by dilution of sucrose solutions. As an example, heat quantities on the order of 10 mJ accompanying the addition of 10 μl titrant solution could be determined with an accuracy of better than 1%. A stepwise titration procedure was used to characterize the binding of indole-3-propionic acid to α-chymotrypsin. The following thermodynamic data were obtained (25°C, acetate buffer, pH 5.80): ΔG⁰ = ?18.46±0.17 kJ·mol^?1, ΔH⁰ = ?15.26±0.20 kJ·mol^?1, ΔS⁰ = 10.85±1.21 JK^?·mol^?1.     

STUDIES IN HISTOCHEMISTRY : LVII. Determination of the Total Dry Mass of Human Erythrocytes by Interference Microscopy and X-ray Microradiography

The total dry mass of human erythrocytes was determined by both interference microscopy and x-ray microradiography. The determination of mass per unit area, and calculation of total dry mass per cell were simplified by changing the shape of the cells to spheres which were then flattened to discs of constant thickness when smeared on glass slides for measurement of fixed cells by interferometry, and to oblate spheroids when smeared on parlodion-coated slides for measurement of fixed cells by x-ray absorption. From x-ray measurements of 100 smeared and alcohol-fixed cells a mean dry mass per cell of 33.7 x 10^-12 g was obtained. Interference measurements of 100 fresh cells suspended in isotonic saline gave a mean value of 32.4 x 10^-12 g while interference measurement of 100 smeared and alcohol-fixed cells gave a mean value of 30.8 x 10^-12 g. The first two values compare well with a mean corpuscular hemoglobin of 31.2 x 10^-12 g, obtained from determinations of erythrocyte count and hemoglobin, since 95 per cent of the dry mass of the cell is hemoglobin. The difference in interference values between the fixed and fresh cells is possibly due to a difference between the specific refractive increment of alcohol-denatured hemoglobin and that of the unmodified substance. The value for the latter was used since that of the former is unknown.     

Sea urchin egg fertilization studied with a fluorescent probe (ANS)

The rates of intracellular DNA synthesis at various temperatures between 39 ° and 31 °C were determined in hamster fibroblasts and HeLa cells by measuring average amounts of ³H-thymidine incorporated per cell in S phase per unit of time. The energy of activation and Q₁₀ for intracellular DNA synthesis were calculated from the slopes of the relative rates of DNA synthesis in HeLa cells and hamster fibroblasts vs. time, plotted on Arrhenius coordinates. In both cell types the incorporation of thymidine into DNA is characterized by an energy of activation of 21 000 calories/mole and a Q₁₀ of 2.94. The absolute rates of DNA synthesis were determined in hamster cells at various temperatures, with values ranging from 1.44 to 0.60 × 10^?14 g DNA/ min/cell at 39 ° to 31 °C, respectively. The length of the S phase of the hamster cell was calculated over a 39 ° to 31 °C range, and found to be 5.0 to 11.9 h, respectively. It is concluded that the S phase length is partly determined by the rate of temperature-dependent DNA synthesis.     

Kinetic analysis of the growth of Spirulina sp. in batch culture

Batch cultivation of Spirulina sp. was carried out under limited light at 30°C in the pH range of 9.2 to 9.7. The specific growth rate D was calculated from the tangent of the growth curve and the cell concentration at that time, and the amount of light energy absorbed per unit time per unit cell weight (E_x), namely, the specific absorption rate of light energy, was also calculated from the total amount of radiant flux of transmitted light at the surface of the culture vessel and cell concentration of the culture solution. A plot against E_x of D in the linear growth phase in batch culture and at various phases in continuous culture gave, for E_x of less than 1.0 kcal/g·h, points scattered near a straight line with slope m 0.037 g/kcal and an intercept on the ordinate, −b, of −0.0046 h⁻¹, and, for higher E_x values, points scattered near a curve of gradually decreasing slope which tended to approach a constant value.A Lineweaver-Burk plot of the reciprocal of D plus b against that of E_x yielded an equation for the growth rate which represented well the growth curve in batch culture. This equation also expressed the linear increase of D with increase of E_x at high cell concentration in the culture solution. The relation between cell growth rate and cell fluidity is discussed by use of a vector equation obtained by applying this relation to a culture solution contained in a given closed surface.     

Evaluation of cardiac energetics by non-invasive 31P magnetic resonance spectroscopy

Alterations in myocardial energy metabolism have been implicated in the pathophysiology of cardiac diseases such as heart failure and diabetic cardiomyopathy. ³¹P magnetic resonance spectroscopy (MRS) is a powerful tool to investigate cardiac energetics non-invasively in vivo, by detecting phosphorus (³¹P)-containing metabolites involved in energy supply and buffering. In this article, we review the historical development of cardiac ³¹P MRS, the readouts used to assess cardiac energetics from ³¹P MRS, and how ³¹P MRS studies have contributed to the understanding of cardiac energy metabolism in heart failure and diabetes.This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Processes involved in the creation of buffering capacity and in substrate-induced proton extrusion in the yeast Saccharomyces cerevisiae

The high pH-maintaining capacity of yeast suspension after glucose-induced acidification, measured as its ability to neutralize added alkali, was found to be due mainly to actively extruded acidity (H⁺). The buffering action of passively excreted metabolites (CO₂, organic acids) and cell surface polyelectrolytes contributed only 15–40% to the overall pH-maintaining capacity which was 10 mmol NaOH/l per pH unit between pH 3 and 4 and 3.5 mmol NaOH/l per pH unit between pH 4 and 7. The buffering capacity of yeast cell-free extract was still higher (up to 4.5-times) than that of glucose-supplied cell suspension; addition of glucose to the extract thus produced considerable titratable acidity but negligible net acidity. The glucose-induced acidification of yeast suspension was stimulated by univalent cations in the sequence $\text{K}{}^{\mathrm{+}}\text{>}\text{Rb}{}^{\mathrm{+}}\text{>>}\text{Li}{}^{\mathrm{+}}\text{～-}\text{Cs}{}^{\mathrm{+}}\text{～-}\text{Na}{}^{\mathrm{+}}$. The processes participating in the acidification and probably also in the creation of extracellular buffering capacity include excretion of CO₂ and organic acids, net extrusion of H⁺ and K⁺ (in K⁺-free media; in K⁺-containing media this is preceded by an initial rapid K⁺ uptake), and movements of some anions (phosphate, chlorides). The overall process appears to be electrically silent.     

Acid Homeostasis Following Partial Ischemia in Neonatal Brain Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

The purpose of this study was to investigate neonatal brain energy metabolism, acid, and lactate homeostasis in the period immediately following partial ischemia. Changes in brain buffering capacity were quantified by measuring mean intracellular brain pH, calculated from the chemical shift of P_i, in response to identical episodes of hypercarbia before and after ischemia. In addition, the relationship between brain buffer base deficit and intracellular pH was compared during and following ischemia. Thus, in vivo ³¹P and ¹H nuclear magnetic resonance spectra were obtained from the brains of seven newborn piglets exposed to sequential episodes of hypercarbia, partial ischemia, and a second episode of hypercarbia in the postischemic recovery period. For the first episode of hypercarbia, brain buffering was similar to values reported for adult animals of other species (percentage pH regulation = 54 ± 16%). During ischemia, the brain base deficit per unit change in pH was ?19 ± 5 mM/pH unit, which is similar to values reported for adult rats. By 20–35 min postischemia, brain acidosis partly resolved in spite of a net increase in lactate concentration. Therefore, the consumption of lactate could not explain acid homeostasis in the first 35 min following ischemia. We conclude that H⁺/HCO^-₃ or other proton equivalent translocation mechanisms must be sufficiently developed in piglet brain to support acid regulation. This is surprising, because a substantial body of evidence implies these processes would be less active in immature brain. The second episode of hypercarbia, from 35 to 65 min postischemia, resulted in a smaller decrease in brain pH compared with the first episode, a result indicating an increase in brain buffering capacity (percentage pH regulation = 79 ± 29%). This was associated with a parallel decrease in brain lactate content, and therefore acid regulation could be attributed to either continued ion translocation or the consumption of lactate. A mild decrease in brain pH and content of energy metabolites was observed, a finding suggesting that the metabolic consequences of severe postischemic hypercarbia are neither particularly dangerous or beneficial.     

The effect of low concentrations of uncouplers on the detectability of proton deposition in thylakoids. Evidence for subcompartmentation and preexisting pH differences in the dark

Flash-induced pH changes inside thylakoids were measured with neutral red as an indicator in the presence and absence of low concentrations of uncouplers. We found that both carrier-type and pore-forming uncouplers caused the rapidly rising phase of the neutral red signal, previously attributed to proton deposition by water oxidation, to disappear. Gramicidin was particularly efficient in this respect, requiring only one molecule of uncoupler per 10⁴ chlorophyll molecules to render the rapid proton deposition undectectable. This suggests that gramicidin did not act on each water-oxidizing enzyme individually, but rather at the level of the thylakoid membrane. In contrast to the effect on water-derived protons, the appearance of protons from plastoquinol was unaffected by gramicidin. Nor did gramicidin affect the rise of the neutral red signal due to proton deposition during two Photosystem I partial reactions with artificial donors. At the low gramicidin concentrations used, its effect on the neutral red signal could not be attributed to a general increase in proton permeability of the thylakoid membrane (acceleration of half decay from 9 to 0.8 s). The extent of alkalinization of the external medium during the first few hundred milliseconds following a light flash was unaltered by gramicidin, and we did not observe a kinetic correlation between the disappearance of the water proton and the decay of the transmembrane electric field. The last two findings suggest that the undetected protons had not crossed the thylakoid membrane, but instead were buffered away by some gramicidin-induced extra buffering power. pH titration of this extra buffering power revealed an apparent pK ranging between 7.2 and 7.7 and a stoichiometry of $\text{2}\text{H}{}^{\mathrm{+}}\text{site}$. The rapid phase of the neutral red signal regained 90% of its original amplitude after seven flashes were applied at 6.7 Hz repetition rate to a sample containing gramicidin. This suggests limits to the extra buffering power. One possible interpretation of our experiments is the following: Protons derived from water oxidation are initially deposited into extended and highly buffering special domains, and only escape into the common internal phase when the buffering capacity of the domains is saturated. As an alternative one may consider that the thylakoid lumen is partitioned into at least two domains, each dominated by different photosystems and with slow proton equilibration between them. Either view requires internal subcompartmentation. The consequences of such subcompartmentation for chloroplast bioenergetics are still obscure.     

Partitioning of Triton X-100, deoxycholate and C10EO8 into bilayers composed of native and hydrogenated egg yolk sphingomyelin

We have used isothermal titration calorimetry (ITC) to study the thermodynamics of Triton X-100 (T_X-100), deoxycholate and decyl octaethylene glycol (C₁₀EO₈) penetration into bilayers composed of native (ESM) and hydrogenated egg yolk sphingomyelin (DHSM). Light scattering measurements were used to study the point of saturation (R_e,sat) and the onset of solubilization of membranes by the detergents. We found that DHSM bilayers at 25 °C were much more resistant to detergent partitioning (lower K) and gave higher reaction enthalpies (ΔH) for all three detergents compared to the ESM bilayer system. Because DHSM lacks double bonds (Δ^4trans and some cis bonds as well), attractive acyl chain interactions are favored in membranes of this lipid class. The high stability and cohesion of DHSM in membranes could be a crucial functional property of this lipid as it is enriched in eye lens membranes.     

Poly-acid properties of biosynthetic hyaluronan studied by titration

In this study the poly-acid properties of biosynthetic hyaluronan produced by fermentation of Bacillus subtilis have been investigated. Potentiometric titration as well as ¹H NMR titration have been used to determine the dissociation constants of the carboxylic group on hyaluronic acid. The intrinsic pK_a and pK_{a, α=0.5} were determined in the presence of 0.1 M salt to be 2.99 and 3.37, respectively. The pK_{a, α=0.5} was found to be unaffected by variations in the ionic strength which is in good agreement with the fact that at α = 0.5, 50% of the carboxylic moieties on the hyaluronan are charged. On the other hand the intrinsic pK_a was found to be dependent on the ionic strength until the Debye-Hückel screening length approaches the length of repeating disaccharide unit of hyaluronic acid.Our findings are in good agreement with previously determined dissociation constants for other sources of hyaluronan. We have also shown that ¹H NMR spectroscopy is the preferred method for polyelectrolyte titration because of the ability to isolate the contribution of several ionisable groups on a polymer on molecular level.     

Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H₂O₂ (JH₂O₂) production. H₂O₂ is reduced to H₂O by electrons supplied by NADPH. NADP⁺ is reduced back to NADPH by activation of mitochondrial membrane potential–dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH₂O₂ production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through β-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP– and low-ADP–stimulated respiration that accelerating flux through β-oxidation generates a corresponding increase in mitochondrial JH₂O₂ production, that the majority (∼70–80%) of H₂O₂ produced is reduced to H₂O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within β-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH₂O₂ production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.     

The effect of size and temperature on the predation of cockles Cerastoderma edule (L.) by the shore crab Carcinus maenas (L.)

Laboratory studies demonstrated that shore crabs Carcinus maenas (L.) can consume <40 cockles Cerastoderma (= Cardium) edule (L.) per individual · day⁻¹. Various predation techniques used by the crabs are reported. The time required to open and consume individual cockles increased exponentially with prey size. Small (<l5-mm shell height), easily broken cockles appeared to be the most profitable in terms of energy acquisition per unit of handling time, the optimal size of prey increasing with predator size. With unlimited prey available, however, crabs selected prey of mean size smaller than these predicted optima, and much below the maximum size they were capable of opening. Feeding rates, both in terms of cockles ingested or energy intake per day, rose steeply with increasing temperature, but the size range of prey consumed remained unchanged. These data strongly suggest that Carcinus maenas is a potentially important predator of small cockles, particularly during the wanner summer months.     

Stabilization of the synthetic media for plant tissue and cell cultures

In standardMurashige-Skoog medium, particularly at pH higher than 5.0 and after heat sterilization, there is a tendency for turbidity or a sediment to appear, and for the acidity to increase by 0.2 to 0.5 degrees pH. The sediment is an amorphous precipitate of ferric phosphate and partly also of ferrous phosphate. In a stock iron solution prepared by chelation of ferrous sulphate with an equimolar quantity of the complexone Na₂EDTA. up to 10% free Fe^II ions could be detected. By titration of a concentrated complexon solution it was found that in the presence of an excess of Na₂EDTA (at the approximate molar ratio Fe^II: Na₂EDTA 1: 2) chelation of this free iron takes place to such an extent that its concentration falls to as little as 0.1%. Media with iron stabilized in this way are quite clear and maintain the adjusted pH for up to several weeks. The heat sterilization, too, does not lead to any precipitation or to a shift in pH within the broad range of adjusted values pH 4.8 – 6.0. We also attempted to increase the relatively low buffering capacity of Murashige-Skoog medium. The addition of sodium citrate (1.25 mmol 1^-1) and particularly of citrate-phosphate buffer (at a final concentration of 1.97 mmol citric acid and 6.07 mmol dibasic sodium phosphate per litre of medium) to the Murashige-Skoog medium considerably increased its buffering capacity, so that at the end of the subculture interval of tobacco cell suspensions the adjusted acidity changed only slightly (pH 5.40 ± 0.15). A thorough evaluation of the growth parameters of tobacco batch cultures (cell counts, vital staining, kinetics of DNA and protein synthesis) failed to reveal any negative effect either of additional chelation or of the buffering components.     

Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca current in liver cells

Uncouplers of mitochondrial oxidative phosphorylation, including carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and carbonilcyanide m-cholorophenylhydrazone (CCCP), are widely used in experimental research to investigate the role of mitochondria in cellular function. Unfortunately, it is very difficult to interpret the results obtained in intact cells using FCCP and CCCP, as these agents not only inhibit mitochondrial potential, but may also affect membrane potential and cell volume. Here we show by whole-cell patch clamping that in primary rat hepatocytes and H4IIE liver cells, FCCP induced large proton currents across the plasma membrane, but did not activate any other observable conductance. In intact hepatocytes FCCP inhibits thapsigargin-activated store-operated Ca²⁺ entry, but in patch clamping under the conditions of strong Ca²⁺ buffering it has no effect on store-operated Ca²⁺ current (I_SOC). These results indicate that there is no direct connection between mitochondria and activation of I_SOC in liver cells and support the notion of indirect regulation of I_SOC by mitochondrial Ca²⁺ buffering.