Net movements of monovalent and bivalent cations, and their relation to energy metabolism, in slices of hepatoma 3924A and of a mammary tumour

1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K⁺ and gained Na⁺, Ca²⁺ and water.

2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K⁺ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca²⁺ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.

3. When respiration was inhibited in the presence of glucose, K⁺ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.

4. In the absence of glucose, the transport of Na⁺ and K⁺ was completely stopped by respiratory inhibition. However, more than 50% of the O₂ uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.

5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.

6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K⁺ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

Studies on the exchange of G-actin-bound calcium with bivalent cations

1. The possibility of the replacement of G-actin-bound calcium by various bivalent cations has been investigated. After the reaction with all cations studied, with the exception of Cu²⁺, action remains active, i.e., contains bound ATP and polymerizes in 0.1 M KCl.

2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin.

3. The rate of exchange is of the same order for bivalent cations studied, including calcium.

4. G-actin-bound Ca²⁺ is fully replaced, besides free Ca²⁺, by free Mn²⁺ and Cd²⁺. The replacement with Mg²⁺, Co²⁺, Ni²⁺ and Zn²⁺ is not complete, and there is practically no reaction with Ba²⁺ and Sr²⁺.

5. Assuming the affinity constant of Ca²⁺ as 1, the following affinity constants for other bivalent cations were obtained: Mn²⁺, 0.90; Cd²⁺, 1.07; Mg²⁺, 0.27; Zn²⁺, 0.22; Co²⁺, 0.18; Ni²⁺, 0.08.

6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca²⁺.     

Reserpine as an uncoupling agent

1. Reserpine, like the uncoupling agent, 2,4-dinitrophenol prevents oxidative phosphorylation but stimulates the rate at which oxygen is reduced.

2. Both reserpine and 2,4-dinitrophenol fail to stimulate oxygen uptake by isolated mitochondria in the presence of arginine.

3. Both 2,4-dinitrophenol and reserpine induce proton permeability in the mitochondrial membrane so that H⁺ is absorbed from the suspending medium.

4. When the reaction system contains reserpine, accumulation of Ca²⁺ by mitochondria is inhibited.

5. Reserpine decreases both ADP:O and P:O ratios which strongly suggest that reserpine is an uncoupling agent.     

Ion transport in heart mitochondria. XIII. The effect of ethylenediaminetetraacetate on monovalent ion uptake

1. Ethylenediaminetetraacetate (EDTA) markedly activates the accumulation of Na⁺ and Li⁺ and the swelling which accompanies the ion uptake by isolated heart mitochondria. This activation is reflected in the removal of limited amounts of endogenous Mg²⁺ and extensive loss of K⁺. The removal of these cations requires the presence of Na⁺, a source of energy, and a permeant anion as well as EDTA. The effects of EDTA on the activation of Na⁺ uptake and cation removal are duplicated by chelators with a high affinity for Mg²⁺, but not by ethyleneglycol-bis-(β-aminoethylether)-N, N′-tetraacetic acid. Mg²⁺ at concentrations 5 to 6 times less than EDTA prevents both activation of Na⁺ uptake and cation removal.

2. EDTA does not appear to be bound by heart mitochondria. At neutral pH the chelator penetrates into the mitochondrial water volume to the same extent as sucrose and mannitol. At pH 8.1 where the removal of mitochondrial Mg²⁺ by EDTA is more effective, EDTA penetrates virtually the entire water volume. This penetration requires the presence of a source of energy, a transported cation such as Na⁺, and a permeant anion. It appears possible that the oscillations in ion uptake and swelling observed in the presence of EDTA at pH 8.1 may be related to the presence of the chelator in the interior compartment under these conditions.     

Control of excitation transfer in photosynthesis V. Correlation of membrane structure to regulation of excitation transfer between two pigment systems in isolated spinach chloroplasts

1. Changes in fluorescence yield of chlorophyll a in isolated chloroplasts have been interpreted by means of regulation of excitation transfer between two pigment systems of photosynthesis^5–7. In order to investigate the relationship between the membrane structure of chloroplasts and the regulation of excitation transfer, changes of light scattering and chlorophyll a fluorescence of isolated spinach chloroplasts were measured upon addition of cations, Mg²⁺ and Na⁺. The cations increased the intensities of both light scattering and fluorescence yield. The changes showed similar time courses and concentration dependences. These facts suggest that modification of membrane structure produced by the cations suppresses the excitation transfer between the two pigment systems.

2. In another case of structural change which is induced by light in the presence of N-methylphenazonium methosulfate, there was little correlation between light-scattering and fluorescence changes.

3. Changes in fluorescence yield induced by the addition of Mg²⁺ were measured in disintegrated chloroplasts and fractionated particles. The effects of Mg²⁺ on fluorescence were observed only in preparations of grana stacks, but not in preparations of stroma lamellae. These findings suggest that the excitation transfer is regulated between the two pigment systems located in the grana thylacoid membranes.     

Inhibition of mitochondrial respiration by loss of intra-mitochondrial K

1. A system is described in which intra-mitochondrial K⁺ concentration can be manipulated by the use of dinitrophenol and valinomycin.

2. As mitochondria lose K⁺ a striking inhibition of O₂ consumption occurs with both succinate and DPN-linked substrates, but not if tetramethyl-p-phenylenediamine + ascorbate serves as substrate. Respiration can be re-activated by adding K⁺ to the medium.

3. Results are discussed in terms of sensitivity of electron transport or substrate dehydrogenation to intra-mitochondrial K⁺ content.     

Osmoadaptation of Thiocapsa roseopersicina OP-1 in batch and continuous culture: Accumulation of K+ and sucrose in response to osmotic stress

Abstract Increasing growth medium NaCl concentration inhibited the growth of Thiocapsa roseopersicina OP-1 due to both an increase in the lag phase of the growth cycle and a reduction in specific growth rate. Addition of 0.05% w/v acetate to the growth medium stimulated growth at all NaCl concentrations, but this stimulation was greatest at supra-optimal NaCl concentrations. Optimal growth under all conditions tested in both batch and continuous culture was recorded at a salt concentration of 0.3 M NaCl. The intracellular concentrations of both K⁺ and sucrose increased linearly with increasing growth medium NaCl concentration indicating as osmoregulatory role for these solutes. Time courses of osmoadaptation in batch culture demonstrated a biphasic response to osmotic stress. The initial phase consisted of a rapid accumulation (within 30 min) of K⁺ from the growth medium. This was followed by a slower synthesis of sucrose which partially replaced intracellular K⁺ during the second phase of osmoadaptation.     

ACCUMULATION OF SULFURIC ACID IN DICTYOTALES (PHAEOPHYCEAE): TAXONOMIC DISTRIBUTION AND ION CHROMATOGRAPHY OF CELL EXTRACTS

Four species in the order Dictyotales ( Dictyopteris latiuscula (Okamura) Okamura, D. prolifera (Okamura) Okamura, D. repens (Okamura) Børgesen, and Spatoglossum crassum J. Tanaka) were found to be highly acidic as in some species of the order Desmarestiales (Phaeophyceae). The pH within their cells, presumably that of the vacuole, was estimated to be 0.5 to 0.9 by pH measurements of their cell extracts in distilled water. However, other species of these genera ( D. divaricata (Okamura) Okamura, D. undulata Holmes, and S. pacificum Yendo) did not show high acidity. Ion chromatography of the cell extracts showed that those species contained high concentrations of SO     within their cells, up to 10 times that in seawater but relatively low Cl⁻. The sum of cations examined (Na⁺, NH     , K⁺, Mg²⁺, Ca²⁺) was significantly lower than that of anions (Cl⁻, Br⁻, NO     , SO     ), and the difference is presumed to represent protons (H⁺), causing the extremely low cell sap pH. Estimated cellular proton concentrations calculated from the pH data roughly agreed with those calculated from differences between the sum of cations and anions and that of anions. Although certain other, nonacidic, dictyotalean species also contained high concentrations of SO     , these species contained high concentrations of Mg²⁺, and the sums of cations and anions were balanced.     

The relation of electron transport and photophosphorylation to conformational changes in chloroplasts

1. The rate of the Hill reaction and photophosphorylation, and the ratio of ATP produced to the electron flow are shown to be strongly dependent on the solute concentration of the medium.

2. A large part, but not all, of the requirement for MgCl₂ or phosphate in photophosphorylation can be replaced by SrCl₂ or other solutes.

3. In two-stage photophosphorylation, solutes are required during the light-activation stage.

4. The presence of solutes causes marked changes in the packed volume of the chloroplasts, and their light-scattering properties. These changes are essentially complete within 1 min.

5. The effectiveness of solutes in enhancing the rate of electron transport and photophosphorylation parallels their effectiveness in inducing conformational changes in chloroplasts.

6. It is suggested that the solutes act by inducing a conformational change of the chloroplast structure which is more optimal for electron transfer and coupled phosphorylation.     

Simultaneous production of two different gel-forming exopolysaccharides by an Alteromonas strain originating from deep sea hydrothermal vents

Exopolysaccharide production by the marine bacterium Alteromonas sp. strain 1644 was shown to be stimulated by restricted growth conditions and was optimized in nitrogen limited fed-batch cultures. Exopolysaccharides were either partly secreted in the medium or stayed firmly cell-associated. The cell-polysaccharide associations could be destroyed by dialysis against distilled water, allowing polysaccharide purification. The chemical and rheological characterization of this last polysaccharide showed that it was different from the secreted polysaccharide that has been previously described (polysaccharide 1644). At low ionic concentration (below 0.03 M whatever the nature of the ions), solutions of this new polysaccharide had very low viscosities. However, at higher ionic concentration, it formed a gel or exhibited in solution at low polymer concentration an unusually high temperature dependent viscosity. This behaviour was also dependent on the nature of the ions and the following sequences for cations and anions were NH4 ⁺ > Mg²⁺ > Na ⁺ > Li⁺ > K⁺ > TMA⁺ and Br⁻ > NO₃⁻ > SO₄²⁻ > Cl⁻ > I⁻ respectively.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Salinity response of a freshwater charophyte, Chara vulgaris

Abstract. Chara vulgaris L. growing in an oligohaline lake was adapted to laboratory conditions and subjected to long-term salinity treatments ranging from 0 to 350 mol m ³ NaCl added to the lake water (40–680 mosmol kg ¹). Osmotic potential and concentration of the main osmotically active solutes (K⁺, Na⁺, Mg²⁺, Cl and sucrose) in the vacuolar sap of the central internodal cells were estimated. C. vulgaris did regulate turgor but incompletely. Turgor decreased from 335 mosmol kg ¹ under control conditions to 52–111 mosmol kg ¹ at 350 mol m ³ NaCl. The enhancement of πⁱ was achieved by increase in both ions and sucrose. Sterile and fertile plants differed in their response to osmotic stress. In sterile plants, the ions accounted for about 87% of the vacuolar osmotic potential. The increase of πⁱ under osmotic stress was exclusively due to an accumulation of Na⁺ and Cl^-. In fertile plants, sucrose accounted for about 35% of πⁱ and ions for about 51% Under osmotic stress, sucrose content increased together with the ionic content of Na⁺ and Cl^-.     

Hyper-osmotic stress induces volume change and calcium transients in chondrocytes by transmembrane, phospholipid, and G-protein pathways

Mechanical compression of cartilage is associated with a rise in the interstitial osmotic pressure, which can alter cell volume and activate volume recovery pathways. One of the early events implicated in regulatory volume changes and mechanotransduction is an increase of intracellular calcium ion ([Ca²⁺]_i). In this study, we tested the hypothesis that osmotic stress initiates intracellular Ca²⁺ signaling in chondrocytes. Using laser scanning microscopy and digital image processing, [Ca²⁺]_i and cell volume were monitored in chondrocytes exposed to hyper-osmotic solutions. Control experiments showed that exposure to hyper-osmotic solution caused significant decreases in cell volume as well as transient increases in [Ca²⁺]_i. The initial peak in [Ca²⁺]_i was generally followed by decaying oscillations. Pretreatment with gadolinium, a non-specific blocker of mechanosensitive ion channels, inhibited this [Ca²⁺]_i increase. Calcium-free media eliminated [Ca²⁺]_i increases in all cases. Pretreatment with U73122, thapsigargin, or heparin (blockers of the inositol phosphate pathway), or pertussis toxin (a blocker of G-proteins) significantly decreased the percentage of cells responding to osmotic stress and nearly abolished all oscillations. Cell volume decreased with hyper-osmotic stress and recovered towards baseline levels throughout the duration of the control experiments. The peak volume change with 550 mOsm osmotic stress, as well as the percent recovery of cell volume, was dependent on [Ca²⁺]_i. These findings indicate that osmotic stress causes significant volume change in chondrocytes and may activate an intracellular second messenger signal by inducing transient increases in [Ca²⁺]_i.     

Effect of excess Cu on the photosynthetic apparatus of runner bean leaves treated at two different growth stages

Variations in water binding strength, water relations and the accumulation of solutes during water stress of three durum wheat ( Triticum durum Desf.) cultivars are reported and discussed. Water binding strength was determined by constructing adsorption isotherms at 5 and 20°C and by calculating the differential enthalpy (ΔH) after van't Hoff.
Reducing sugars, proline, K⁺ and Cl⁻ were the major contributors to osmotic adjustment. Solutes, such as quaternary ammonium compounds, and non-reducing sugars contributed to osmotic potential at full turgor, but did not increase in proportion to water stress. Genotypic differences have been observed for K⁺ accumulation capability, the water-stressed leaves of cv. Capeiti 8 showing the largest increase. The same cultivar demonstrated the most negative ΔH values, indicative of strongly bound water, and the highest integrated enthalpy (ΔH_inf) values for leaf moisture below 0.1 g H₂O per g dry weight, i.e. in the isotherm region where water was presumably chemisorbed to the charged groups of macromolecules. The accumulation of ions (Cl⁻, K⁺) and proline was concurrent with an increase in the binding-strength of tightly and weakly bound water, respectively.     

Salt requirements for membrane transport and solute retention in some moderate halophiles

Abstract Many species of bacteria isolated from saline environments require Na⁺ specifically for membrane transport. Transport occurs by a Na⁺ symport process energized by an electrochemical gradient of Na⁺ ions. The gradient at neutral pH appears to be produced by a primary electrogenic extrusion of protons coupled to a secondary, outwardly directed Na⁺ pump, a Na⁺/proton antiporter. At alkaline pH Vibrio alginolyticus may also produce the gradient by an energy-dependent primary extrusion of Na⁺ ions. Alteromonas haloplanktis and Vibrio costicola require salts in the medium to retain intracellular solutes. For A. haloplanktis the effects of the salts are primarily osmotic. For V. costicola , only NaCl is effective in retaining solutes and Na⁺ is required by this organism to maintain the membrane potential. In Escherichia coli a single substitution in the nucleotide sequence of the gene coding for the melibiose transport protein changed the cation specificity of the transport system. The possible ecological significance of this finding has been considered.     

Response to osmotic medium and fusicoccin by seeds of radish (Raphanus sativus) in the early phase of germination

The effect of increasing osmotic values of the medium (mannitol) on the growth and the response mechanisms of seeds of radish ( Raphanus sativus L., cv. Ton do Rosso Quarantino) during the early phase of germination was investigated in the presence or absence of fusicoccin (FC). Decreasing the water potential in the medium inhibited the growth and the evolution of protein synthesis and enhanced H⁺ extrusion, net uptake of K⁺ and malic acid synthesis. FC, which stimulates these latter functions, counteracted the inhibitory effect of the decreasing water potential of the medium on growth and protein synthesis. Neither in the absence nor in the presence of FC did decreasing water potential of the medium enhance the synthesis of soluble sugars and amino acids to support the osmotic pressure of the seeds. The osmotic and water potentials of the seeds increased during germination. FC made the increase more rapid, while mannitol kept both potentials low. The pressure potentials of the seeds also decreased with time, and both FC and mannitol enhanced this change. If the seeds were without turgor, the development of protein synthesis was blocked. The seeds counteract the effect of decreasing water potentials in the medium by: a) enhancing H⁺ extrusion (and, as a consequence, wall loosening and transport mechanisms) and the synthesis of malic acid as apparent in the presence of FC; b) regulating the osmotic potentials of the cells (with a lower dilution of the osmotic compounds present in the seeds due to the diminished uptake of water); c) controlling the growth through the effects of a) and b) on the pressure potentials (internal hydrostatic pressure) of the seeds and on protein synthesis.     

A relationship between potassium and proline accumulation in salt-stressed Sorghum bicolor

The amount of total monovalent cations in leaves of Sorghum bicolor , L. Moench, RS 610, which were exposed to salinity stress, was a function of both the osmotic potential and the concentration of K⁺ of growth media. The plants have a Na⁺ exclusion mechanism that keeps the level of Na⁺ in leaves low. Thus, most of the osmotic adjustment in leaves was due to K⁺. Proline did not start to accumulate in leaves until the concentration of total monovalent cations in leaves reached a threshold of approximately 200 μmol/g fresh weight. Above this threshold, the contents of prolioe and monovalent cations in leaves increased with increasing salinity of the medium. The ratio of proline to monovalent cation was 5% of that amount of monovalent cation in excess of the threshold concentration. Therefore, if the cations are located in the vacuoles and proline accumulates in the cytoplasm, then the amount of accumulated proline is sufficient to act as a balancing osmoticum across the tonoplast. Very little proline accumulated in roots because this tissue contained much less total monovalent cations than leaves from the same salt-stressed plants. The same threshold of 200 μmol/g fresh weight of total monovalent cations was required in roots as in leaves to initiate proline accumulation.     

Uptake of metals by bacterial polysaccharides

J.L. GEDDIE AND I.W. SUTHERLAND. 1993. The binding of cations by a range of bacterial polysaccharides was examined. Comparison of native and deacetylated polymers indicated the influence of polysaccharide acetylation on ion uptake and selectivity. The effects of temperature and pH on ion uptake were also examined. Metal ion uptake was carried out by dialysis and samples were analysed using ion chromatography. The native acetylated polymers showed a selectivity for Ca²⁺ > Mg²⁺ > monovalent cations, whereas samples lacking acetyl groups showed a selectivity for monovalent cations > Mg²⁺ > Ca²⁺. Increased temperatures reduced the capacity for several of the polymers to bind the cations; The Zoogloea ramigera polymer appeared least affected. The pH value also affected uptake.     

Oxidative phosphorylation in Azotobacter vinelandii. Effect of inhibitors and uncouplers on P/O ratio, trypsin-induced ATPase and ADP-stimulated respiration

1. The effect of various inhibitors and uncouplers of mitochondrial oxidative phosphorylation on phosphorylation by Azotobacter particles is reported.

2. The ATPase activity of Azotobacter particles and of the soluble factor involved in oxidative phosphorylation is stimulated 10–20-fold by incubation with trypsin. The trypsin-induced ATPase is Mg²⁺ or Ca²⁺ dependent, Mg²⁺ being the more effective cation. The ATPase is stimulated somewhat by 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, desaspidin and carbonyl cyandie m-chlorophenylhydrazone, and inhibited by atebrin and Dio-9.

3. In previous work ADP was shown to stimulate the oxidation of NADH but not of the Site-II substrates malate, lactate or succinate. Stimulation by ADP of oxidation of malate has now been observed by inhibiting electron transport by 2-heptyl-4-hydroxyquinoline-N-oxide or by increasing the electron flow by adding lactate. The stimulation is, therefore, not confined to phosphorylation Site I.