The monensin-mediated transport of Na+ and K+ through phospholipid bilayers studied by 23Na- and 39K-NMR

Addition of monesin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine (EPC) in sodium or potassium chloride solution and from dioleoylphosphatidylcholine (DOPC) in sodium chloride solutions gives rise to dynamic 23Na- and 39K-NMR spectra. The dynamic spectra arise from the monensin-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of monensin and metal ion concentrations and are compatible with a model in which one monensin molecule transports one metal ion. Rate constants for the association and dissociation of the monensin-metal complex in the membrane/water interface are extracted and the stability constants for complex formation are evaluated. The rate constants in DOPC are similar to those in EPC, confirming that diffusion is not rate-limiting in the transport process and that dissociation of the complex is the rate-limiting step. Although potassium on its own is transported more rapidly, sodium forms the more stable complex and is therefore transported preferentially in competition with potassium.     

The specific requirement for sodium chloride for the active uptake of l-glutamate by Halobacterium salinarium

1. Uptake of l-glutamate by Halobacterium salinarium is dependent on high concentrations of sodium chloride in the environment. When the sodium chloride is replaced by isomolar concentrations of potassium chloride, sodium acetate or potassium acetate, only negligible uptake occurs. 2. Most of the glutamate taken up can be shown to be in the cells in the free state and at a concentration of at least 50 times that in the medium. Sodium chloride is therefore required for an active transport of the glutamate into the cells. 3. The question whether sodium chloride is essential for the actual migration of glutamate across the cell envelope or for the mechanism supplying energy for this migration is discussed on the basis of experiments on endogenous respiration and with inhibitors.     

Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel

Summary A potassium-specific tonoplast channel was identified by reconstitution of tonoplast polypeptides into planar lipid bilayer membranes. Highly purified tonoplast membranes were solubilized in Triton X-100-containing buffer and fractionated by size-exclusion chromatography. The protein fractions were assayed for ion channel activity in a planar bilayer system, and the potassium channel was routinely recovered in specific fractions corresponding to an apparent molecular mass of 80 kDa. In symmetrical electrolyte solutions of 100 mM potassium chloride, the potassium channel had a single-channel conductance of 72 pS. Substates of the channel with conductances of 17, 33 and 52 pS were frequently observed. After identification of the channel in low or high KCl, addition of sodium acetate or sodium chloride caused only insignificant conductance changes. This result suggested that the channel was not or little permeable for sodium or chloride, whereas it had similar single-channel conductance for rubidium and caesium ions as compared with potassium ions. The channel is presumably responsible for the equilibration of potassium between the vacuole and the cytosol. The role of the channel in the physiology of the barley cell under salt stress is discussed.The authors would like to thank U. Heber for many helpful discussions. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 176, projects B3 and B7) and by the Fonds der Chemischen Industrie.     

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.     

On the Composition of “Viscol” and the Employment of Gum Arabic for Mounting Media

“Viscol”, a water soluble permanent mounting medium hardening through evaporation of water under the cover glass, has been analyzed. It proves to consist of a mixture of gum arabic, glycerol, phenol and water and is especially suitable for simple botanical preparations. The use of gum arabic for hardening permanent mounting media is reviewed. Instead of a glycerol-phenol-water mixture a lactophenol, potassium acetate or zinc chloride solution mixed with gum arabic may be used for a permanent mounting medium. However, gum arabic contains calcium, magnesium and potassium ions giving crystals with the solutions mentioned. In the case of lactophenol and potassium acetate, the calcium and magnesium ions must be removed beforehand, which is done by precipitation with sodium or potassium carbonate. In the case of zinc chloride the potassium ions must be removed, which is done by dialysis with zinc chloride. It is pointed out that the same principles may be used for a great variety of different mounting media.     

The biology of simocephalus acutirostratus king (Cladocera: Daphnidae)-hatchability of the parthenogenetic egg cultured in artificial media

The development of the parthenogenetic egg of the daphnid, Simocephalus acutirostratus was studied in different artificial media. The hatchability is found to be 100% in 0.001 M to 0.01 M of sodium chloride. 100% hatchability is noticed in concentrations ranging from 0.001 M to 0.05 M of calcium chloride. Apart from minor fluctuations, a high percentage of hatchability is recorded when isotonic solutions of sodium and calcium chloride are mixed. The hatchability of the different stages of the embryo is studied in an artificial medium which contains various proportions of sodium chloride, potassium chloride and calcium chloride. The results are discussed and compared with those from an allied temperate form, Simocephalus vetulus (O. F. Muller).     

Ionophore characteristics of heliomycin]

A highly purified product, identified as a well-known antibiotic geliomycin, has been derived by gradient extraction of the mycelium from Streptomyces robefuscus isolated from soil specimens as a result of purposeful search for a producer of hydrophobic ionophore antibiotics. The ionophore properties of geliomycin are highly labile and inducible. Cationic or anionic nature and the angle of the function performed depend on the concentration, type of electrolyte, protocol of measurements, and some additives used for the preparation of geliomycin electrodes to measurements. Stable development of the cationic function was detected for potassium chloride. The lability of ionophore properties was the highest with ammonium chloride as the electrolyte. Ammonium acetate and potassium and sodium chlorides notably increased the stability of geliomycin electrodes. Selective activity of geliomycin towards potassium ion is a more permanent characteristic. The results permit a conclusion about the inducibility of ionophore properties of geliomycin, which may play an important role in the vital activity of the producer and manifest as a regulatory factor in maintenance of water and acid base balance.     

A potential anti-oxidant protein in a ferrous iron-oxidizing Sulfolobus species

Abstract Eight species of halophilic Archaea were tested for the presence of isocitrate lyase activity. High activities (up to 100 nmol min⁻¹ mg protein⁻¹) were detected in Haloferax mediterranei and Haloferax volcanii when grown in medium containing acetate as the principal carbon source. Little activity was found in representatives of the genera Halobacterium and Haloarcula . Isocitrate lyase from Haloferax mediterranei required high potassium chloride concentrations, optimal activity being found at 1.5–3 M potassium chloride and pH 7.0. Replacement of potassium chloride by sodium chloride resulted in much lower activities. Sulfhydryl compounds (cysteine, glutathione) were not stimulatory. In other properties (stimulation by magnesium ions, sensitivity to different inhibitors) the enzyme resembled isocitrate lyases from representatives of the Bacteria and Eucarya.     

THE IONIZATION OF PROTEIN CHLORIDES

1. By the use of the silver-silver chloride electrode, measurements have been made of the chloride ion concentrations of 1 per cent solutions of five proteins, containing from 0.001 N to 0.1 N hydrochloric acid. The hydrogen ion concentrations of the same solutions have been measured by the use of the hydrogen electrode. 2. The measurements indicate that the chlorides of gelatin, egg albumin, casein, edestin, and serum globulin are highly ionized electrolytes, ionizing to yield chloride ion and a positive protein-hydrogen ion. Their ionization is therefore similar to that of ammonium chloride. 3. The results do not support the idea that a protein chloride does not yield chloride ion on dissociation. They are not in agreement with the idea that the depressing effect of an excess of HCl on the viscosity and other colloidal properties of a protein chloride solution is due to a repression of the ionization of the protein chloride. The results are, however, in complete accord with the theory of colloidal behavior advocated by Loeb.     

The initiation of cell division in theChaetopterus Egg

Summary The various agents which can cause the initiation of cell division in the egg of the wormChaetopterus all cause an increase in the viscosity of the interior protoplasm. These agents include hypertonic solutions, isotonic potassium chloride solutions, cold, heat, acid, alkali, ether and ultraviolet radiation.This investigation was supported by a research grant from the National Cancer Institute, National Institutes of Health, Public Health Service.     

Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica

Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.     

Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.     

The localization of transport properties in the frog lens.

The selectivity of fiber-cell membranes and surface-cell membranes in the frog lens is examined using a combination of ion substitutions and impedance studies. We replace bath sodium and chloride, one at a time, with less permeant substitute ions and we increase bath potassium at the expense of sodium. We then record the time course and steady-state value of the intracellular potential. Once a new steady state has been reached, we perform a small signal-frequency-domain impedance study. The impedance study allows us to separately determine the values of inner fiber-cell membrane conductance and surface-cell membrane conductance. If a membrane is permeable to a particular ion, we presume that the conductance of that membrane will change with the concentration of the permeant ion. Thus, the impedance studies allow us to localize the site of permeability to inner or surface membranes. Similarly, the time course of the change in intracellular potential will be rapid if surface membranes are the site of permeation whereas it will be slow if the new solution has to diffuse into the intercellular space to cause voltage changes. Lastly, the value of steady-state voltage change provides an estimate of the lens' permeability, at least for chloride and potassium. The results for sodium are complex and not well understood. From the above studies we conclude: (a) surface membranes are dominated by potassium permeability; (b) inner fiber-cell membranes are permeable to sodium and chloride, in approximately equal amounts; and (c) inner fiber-cell membranes have a rather small permeability to potassium.     

Advantages of rubidium chloride gradients for isopycnic centrifugation of enzymes

The isopycnic centrifugation of beta-amylase and the marker enzyme beta-galactosidase was carried out using four salts viz. rubidium chloride, potassium bromide, potassium acetate and lithium bromide. Lithium bromide inactivated both beta-galactosidase and beta-amylase. The high viscosity of potassium acetate gradients necessitated an extremely long centrifugation time. The density profiles obtained with rubidium chloride gradients were sharper and permitted better resolution than potassium bromide gradients. Both enzymes were stable in rubidium chloride gradients, while potassium bromide inactivated beta-galactosidase, even in the presence of 10 mM 2-mercaptoethanol.     

Salinity tolerance of Gambusia affinis (Baird & Girard) and Heteropneustes fossilis (Bloch)

The present paper describes the results of a study on the salinity tolerance of two freshwater fishes, Gambusia affinis (Baird & Girard) and Heteropneustes fossilis (Bloch). The two species were treated with various concentrations of sea water and three most common salts found in the saline waters of Iraq, namely sodium chloride, calcium chloride and potassium chloride. The mortality rates following these treatments were recorded. Both species showed the highest degree of resistance for sea water and sodium chloride, and the lowest degree of tolerance to potassium chloride. Gambusia affinis was more resistant to these salts and sea water than H. fossilis .     

The Control of Sea Urchin Metamorphosis: Ionic Effects

Because the cascade of events which comprise sea urchin metamorphosis occur rapidly, regulatory mechanisms able to respond in minutes must function. Employing sea water solutions of altered ionic composition in the presence or absence of metamorphically active microbial films, we tested the ability of particular ions to inhibit or enhance metamorphosis in competent larvae of the sea urchin, Lytechinus variegatus . At 40 mM excess potassium maximally induces normal metamorphosis in the absence of a microbial film. In the presence of metamorphically active microbial films, 40 mM excess magnesium inhibits the process. Increasing concentrations of calcium up to an excess of 40 mM stimulates larvae to undergo metamorphosis but in smaller proportions than similar concentrations of potassium. Divalent cation-free sea water solutions are toxic to larvae. These studies support the hypothesis that ion fluxes are involved in the regulation of metamorphosis and reveal a complexity of response that parallels the histological complexity of competent echinoid larvae.     

Salt excretion by the perfused head of trout adapted to sea water and its inhibition by adrenaline

Summary Isolated heads of trout (Salmo gairdneri) were used to study the unidirectional flux of sodium and chloride across the gills in salt water.Two perfusion techniques were employed. Under constant pressure perfusion, the addition of adrenaline during the perfusion causes an increase in the flow-rate. Under constant flow-rate conditions, adrenaline provokes a decrease in pressure. A comparison of influx determination made with these two techniques of perfusion shows that variations in flow-rate of perfusion do not affect the assessment of these fluxes.A net efflux of sodium, but not of chloride, is demonstrated in sea water. The effluxes of sodium and chloride observed in sea water are decreasedd about 50% during a rapid transfer to fresh water. The addition of potassium to this medium stimulates the effluxes of sodium and chloride, suggesting a Na/K exchange participating in the chloride excretion.Adrenaline causes an inhibition of sodium and chloride efflux in sea water which persists after transfer to fresh water and the addition of potassium. Only the influx of chloride is inhibited at a concentration of 10^–5 M whereas the sodium influx is unaffected. The presence of adrenaline results in a net influx of both sodium and chloride.The differential action of adrenaline on the influxes of sodium and chloride suggests that the hemodynamic modifications provoked by this catecholamine occur independently of its aforementioned ion exchange effects.     

THE COLLOIDAL BEHAVIOR OF EDESTIN

1. It has been shown by titration experiments that the globulin edestin behaves like an amphoteric electrolyte, reacting stoichiometrically with acids and bases. 2. The potential difference developed between a solution of edestin chloride or acetate separated by a collodion membrane from an acid solution free from protein was found to be influenced by salt concentration and hydrogen ion concentration in the way predicted by Donnan''s theory of membrane equilibrium. 3. The osmotic pressure of such edestin-acid salt solutions was found to be influenced by salt concentration and by hydrogen ion concentration in the same way as is the potential difference. 4. The colloidal behavior of edestin is thus completely analogous to that observed by Loeb with gelatin, casein, and egg albumin, and may be explained by Loeb''s theory of colloidal behavior, which is based on the idea that proteins react stoichiometrically as amphoteric electrolytes and on Donnan''s theory of membrane equilibrium.     

ELECTROKINETIC PHENOMENA : XI. ACTION OF UNI-UNIVALENT ELECTROLYTES ON ELECTRIC MOBILITY OF PROTEINS

By assuming that the electric charge of proteins is primarily determined by the hydrogen ion activity of the medium, and by making corrections when necessary for the effect of salt, it is possible to derive a simple relationship between the electric mobility of proteins and the effective reciprocal thickness of the electric double layer. The decrease in electric mobility of proteins in solutions is readily predicted for gelatin in acetate buffers and for egg albumin in the presence of the alkali halides, of acetate buffers, and of hydrochloric acid on the basis of the assumptions made.     

Effet de divers sels alcalins sur la croissance et la nutrition minérale de Lemna minor L

Lemna minor L. was grown in aseptic culture solutions. With sodium chloride or lithium chloride in the solution, the plant increased in yield. Tolerance of Lemna minor to relatively high level of sodium explains the presence of this plant in brackish waters. A low quantity of potassium is required for optimal growth. Sodium was added to nutrient solutions as chloride or sulphate. At equivalent levels the sodium content of the plant was somewhat higher under sulphate salinity.