Species-dependent differences in the effect of ionic strength on potassium transport of erythrocytes: the role of lipid composition.

The Rb+(K+) efflux of erythrocytes from six mammalian species was investigated in solutions of physiological and low ionic strength. A species dependent increase of the Rb+(K+) efflux in low ionic strength solution could be observed. The rate constant of Rb+(K+) efflux of erythrocytes in physiological ionic strength solution correlates with the content of arachidonic acid of the membrane phospholipids. The same relation was observed in solution of low ionic strength with the exception of human erythrocytes. In addition, an age-dependent correlation of the rate constant of Rb+(K+) efflux from calf erythrocytes in low ionic strength solution with the content of arachidonic acid of the membrane phospholipids was found. The Rb+(K+) efflux of human erythrocytes, which is enhanced in low ionic strength solution, decreases with the decreasing temperature. The temperature-dependent ESR order parameter of a fatty acid spin label for human and cow erythrocytes in solution of physiological and low ionic strength media suggested that the effect of low ionic strength on Rb+(K+) efflux is not solely based on a change of membrane fluidity. The results are interpreted as being due to a specific influence of membrane phospholipids on the Rb+(K+) efflux.     

An Analysis of the Surface Fixed-Charge Theory of the Squid Giant Axon Membrane

The observed shift in threshold potential, after perfusion of the squid giant axon with solutions of low ionic strength, can be predicted by assuming a fixed negative charge on the inside of the membrane. The constant field equation, together with the double-layer potential due to this charge, has been used to determine the change in resting potential during perfusion with solutions of low ionic strength. Neither the modified constant field equation nor Planck's diffusion equation can successfully predict the observed shift in resting potential. It is suggested that a positive charge distribution exists about the sodium channel on the outside of the membrane. The double-layer potential due to this positive charge, together with the independence principle, has been used to predict the relationship between sodium current and membrane potential when the ionic strength and sodium activity of the external solution are decreased. These predictions have been compared with the available experimental observations.     

Monte Carlo simulations of supercoiled DNAs confined to a plane.

Recent advances in atomic force microscopy (AFM) have enabled researchers to obtain images of supercoiled DNAs deposited on mica surfaces in buffered aqueous milieux. Confining a supercoiled DNA to a plane greatly restricts its configurational freedom, and could conceivably alter certain structural properties, such as its twist and writhe. A program that was originally written to perform Monte Carlo simulations of supercoiled DNAs in solution was modified to include a surface potential. This potential flattens the DNAs to simulate the effect of deposition on a surface. We have simulated transfers of a 3760-basepair supercoiled DNA from solution to a surface in both 161 and 10 mM ionic strength. In both cases, the geometric and thermodynamic properties of the supercoiled DNAs on the surface differ significantly from the corresponding quantities in solution. At 161 mM ionic strength, the writhe/twist ratio is 1.20-1.33 times larger for DNAs on the surface than for DNAs in solution and significant differences in the radii of gyration are also observed. Simulated surface structures in 161 mM ionic strength closely resemble those observed by AFM. Simulated surface structures in 10 mM ionic strength are similar to a minority of the structures observed by AFM, but differ from the majority of such structures for unknown reasons. In 161 mM ionic strength, the internal energy (excluding the surface potential) decreases substantially as the DNA is confined to the surface. Evidently, supercoiled DNAs in solution are typically deformed farther from the minimum energy configuration than are the corresponding surface-confined DNAs. Nevertheless, the work (Delta A(int)) done on the internal coordinates, which include uniform rotations at constant configuration, during the transfer is positive and 2.6-fold larger than the decrease in internal energy. The corresponding entropy change is negative, and its contribution to Delta A(int) is positive and exceeds the decrease in internal energy by 3.6 fold. The work done on the internal coordinates during the solution-to-surface transfer is directed primarily toward reducing their entropy. Evidently, the number of configurations available to the more deformed solution DNA is vastly greater than for the less deformed surface-confined DNA.     

Interactions des ions métalliques avec le DNA. I. Comportement de polyélectrolyte et liaison des ions compensateurs

The theory of polyelectrolyte solution of Alexandrowicz: and Katchalsky is used to calculate the electrostatic potential of single stranded polynucleotides for different ionic strength. We have considered the potential of double stranded DNA as the superposition of the different potentials produced by each chain, provided the average distance between the strands is higher than an ionic strength-dependent parameter b. For ionic strength lower than 5 × 10^?2M, the assumption is no longer valid, and a cylindrical model with a uniform charge density must be used. The continuity between the two models was tested, and thus we can calculate the electrical potential at the vicinity of a phosphate group in the whole range of ionic-strength where the double helix is stable. It was therefore possible to determine the theoretical number of ions bound electrostatically to DNA and we found an increase of ion binding with a decrease of ionic strength. Such a model was further applied to the change of specific volume in different salt solutions. Comparison is made with recent pycnometric data on Na^? and Cs^? salts of DNA. Agreement is good in the case of Cs⁺, but for Na⁺, cation binding is likely to be accompanied by a change of the hydration of DNA, which depends on ionic strength. With the same model we can see easily the ion-trapping properties of DNA which play an important role in any formation of complex between heavy ions and bases.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Intrinsic electric fields and membrane bending

The fragmentation of human erythrocytes heated in a range of ionic environments has been examined by video microscopy, , the average number of surface wave crests growing on the cell rim during fragmentation by membrane externalization, andI, the percentage of cells internalizing membrane, were scored.The membrane diffusion potential was altered experimentally on decreasing the extracellular chloride concentration by substituting either membrane-impermeant sorbitol or Na gluconate for some NaCl. The external-membrane-face surface potential was altered either by surface charge depletion or by ionic strength changes. The dependence of morphological change on diffusion potential at constant cell volume and surface potentials was established over a 34-mV change in diffusion potential. The rate constants for morphological change with charge depletion at different diffusion potentials are largely independent of the diffusion potential. A l.O-mV increase in diffusion potential has an effect on morphological change of comparable magnitude to that of a 1.0-mV decrease in the modulus of the negative surface potential. When the diffusion potential increased on decreasing both the extracellular diffusible ion concentration and extracellular ionic strength, the effect on cell morphology of increasing the modulus of the surface potential was overcome by the effects of the diffusion potential change.     

The effect of composition and ionic strength of external solution on the aspartate-ammonia lyase and fumarate hydratase activity in Escherichia coli cells

It was found that the nonspecific effect of ionic strength of the external solution on the enzymatic activity of E. coli cells consists in rapid changes in the permeability of cell membranes interacting with the substrate. This effect depends on the initial substrate concentration, i.e., ionic strength of the external solution, and is maintained for some time as the substrate concentration decreases. Chloramphenicol, a protein synthesis inhibitor, and sodium azide, a respiration inhibitor (300 micrograms/ml and 200 microM, respectively) do not change the enzymatic activity of E. coli cells during the synthesis of L-aspartic and L-malic acids from fumaric acid. The kinetic equations of L-aspartate and L-malate synthesis are described by equations of zero and intermediate (between zero and first) order, respectively.     

Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes

DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R(ct) that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R(ct) change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R(ct) change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R(ct) upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.     

Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution

Melittin is a cationic, amphipathic, hemolytic peptide composed of 26 amino acid residues. It is intrinsically fluorescent due to the presence of a single tryptophan residue, which has been shown to be crucial for its hemolytic activity. It undergoes a structural transition from a random coil monomer to an alpha-helical tetramer at high ionic strength. Although the aggregation behavior of melittin in solution is well characterized, dynamic information associated with the aggregation of melittin is lacking. In this paper, we have monitored the effect of ionic strength on the dynamics and aggregation behavior of melittin in aqueous solution by utilizing sensitive fluorescence approaches, which include the red edge excitation shift (REES) approach. Importantly, we demonstrate that REES is sensitive to the self-association of melittin induced by ionic strength. The change in environment experienced by melittin tryptophan(s) is supported by changes in fluorescence emission maximum, polarization, and lifetime. In addition, the accessibility of the tryptophan residue was probed by fluorescence quenching experiments using acrylamide and trichloroethanol as soluble and hydrophobic quenchers, respectively. Circular dichroism studies confirm the ionic strength-induced change in the secondary structure of melittin. Taken together, these results constitute the first report showing that REES could be used as a sensitive tool to monitor the aggregation behavior of melittin in particular and other proteins and peptides in general.     

Effect of external factors on the kinetic properties of PGH-synthase

The influence of external factors, viz. reaction mixture temperature, presence of low- and high-molecular (tween-20) organic compounds, urea and the solution high ionic strength on kinetic properties of PGH synthetase has been studied. Some factors (high ionic strength, addition of organic compounds) were found to slow down irreversible inactivation of PGH synthetase in the course of the reaction. Sensitivity of the enzyme to the change of the solution ionic strength depends on environmental factors, rising on the microsomal enzyme solubilization with tween-20 and decreasing on further enhancement of the tween-20 concentration.     

Generation of the streaming potential by liposomes in cylindrical capillary. Experimental data

A model of creation a streaming potential U as a result of colloidal particle movement in flow in a capillary has been described previously (Zawada 1996) as well as the systems for measurement (Zawada 1990, 1991). The filling of capillary with a solution of liposomes results in a labile adsorbance of liposomes on a capillary glass and changes the measured streaming potential. In order to minimalize these adverse effects, the capillary was covered with phospholipid layer of different composition. Some concentrations of stearylamine as a component of the phospholipid layer may fully compensate the surface charge of the glass capillary and can reduce the liposomes adsorption. The streaming potential of the liposomes solution depends on the ionic strength of the electrolyte and is smaller than the zeta potential for similar liposomes. This suggests that only a part of ions of the liposome ion atmosphere participate in creating of the streaming potential. These are the ions from the hydrodynamic slipping layer. The regression analysis of the relationships between streaming potential U and concentration of liposomes and next ionic strength of the electrolyte gave the value of the surface potential psi0 and the thickness of the hydrodynamic slipping layer d, that is independent of the ionic strength.     

Excessive counterion condensation on immobilized ssDNA in solutions of high ionic strength

We present experiments on the bias-induced release of immobilized, single-stranded (ss) 24-mer oligonucleotides from Au-surfaces into electrolyte solutions of varying ionic strength. Desorption is evidenced by fluorescence measurements of dye-labeled ssDNA. Electrostatic interactions between adsorbed ssDNA and the Au-surface are investigated with respect to 1), a variation of the bias potential applied to the Au-electrode; and 2), the screening effect of the electrolyte solution. For the latter, the concentration of monovalent salt in solution is varied from 3 to 1600 mM. We find that the strength of electric interaction is predominantly determined by the effective charge of the ssDNA itself and that the release of DNA mainly occurs before the electrochemical double layer has been established at the electrolyte/Au interface. In agreement with Manning's condensation theory, the measured desorption efficiency (etarel) stays constant over a wide range of salt concentrations; however, as the Debye length is reduced below a value comparable to the axial charge spacing of the DNA, etarel decreases substantially. We assign this effect to excessive counterion condensation on the DNA in solutions of high ionic strength. In addition, the relative translational diffusion coefficient of ssDNA in solution is evaluated for different salt concentrations.     

Effect of ionic strength on the organization and dynamics of membrane-bound melittin

Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. We have previously shown that the sole tryptophan of melittin is localized in a motionally restricted environment in the membrane interface. We have monitored the effect of ionic strength on the organization and dynamics of membrane-bound melittin utilizing fluorescence and circular dichroism (CD) spectroscopic approaches. Our results show that red edge excitation shift (REES) of melittin bound to membranes is sensitive to the change in ionic strength of the medium. This could be attributed to a change in the immediate environment around melittin tryptophan with increasing ionic strength due to differential solvation of ions. Interestingly, the rotational mobility of melittin does not appear to be affected with change in ionic strength. In addition, fluorescence parameters such as lifetime and acrylamide quenching of melittin indicate an increase in water penetration in the membrane interface upon increasing ionic strength. Our results suggest that the solvent dynamics and water penetration in the interfacial region of the membranes are significantly affected at physiologically relevant ionic strength. These results assume significance in the overall context of the influence of ionic strength in the organization and dynamics of membrane proteins and membrane-active peptides.     

Ionic strength has a greater effect than does transmembrane electric potential difference on permeation of tryptamine and indoleacetic acid across Caco-2 cells

The effects of transmembrane electric potential difference and ionic strength on the permeation of tryptamine and indoleacetic acid across a Caco-2 cell monolayer were examined. A decrease in the transmembrane electric potential difference caused by the addition of potassium ion to the transport buffer had no effect on the permeation rate of either compound. On the other hand, an increase in ionic strength resulted in a decrease in the permeation rate of tryptamine and an increase in the permeation rate of indoleacetic acid. The changes in the permeation rate with changes in the ionic strength were correlated with the membrane surface potential monitored by 1-anilino-8-naphthalenesulfonic acid (ANS), a fluorescent probe. We tested these effects using several other cationic and anionic compounds. These effects of ionic strength were found to be common to all drugs tested. The compound that showed a relatively lower permeation rate was given relatively stronger effect. The possibility of overestimation or underestimation caused by these effects should be considered when the permeation of an ionic compound is evaluated using a cell monolayer system.     

Ionic strength has a greater effect than does transmembrane electric potential difference on permeation of tryptamine and indoleacetic acid across Caco-2 cells

The effects of transmembrane electric potential difference and ionic strength on the permeation of tryptamine and indoleacetic acid across a Caco-2 cell monolayer were examined. A decrease in the transmembrane electric potential difference caused by the addition of potassium ion to the transport buffer had no effect on the permeation rate of either compound. On the other hand, an increase in ionic strength resulted in a decrease in the permeation rate of tryptamine and an increase in the permeation rate of indoleacetic acid. The changes in the permeation rate with changes in the ionic strength were correlated with the membrane surface potential monitored by 1-anilino-8-naphthalenesulfonic acid (ANS), a fluorescent probe. We tested these effects using several other cationic and anionic compounds. These effects of ionic strength were found to be common to all drugs tested. The compound that showed a relatively lower permeation rate was given relatively stronger effect. The possibility of overestimation or underestimation caused by these effects should be considered when the permeation of an ionic compound is evaluated using a cell monolayer system.     

A method for investigating the effect of temperature on the 695 nm band of insoluble cytochrome c.

A method is described for computer analysis of simple spectrophotometric changes in particulate systems, and this has been applied to the bleaching of the 695 nm band of insoluble ferricytochrome c by temperature. The results show that insolubilization has no effect on the standard enthalpy change but lowers the value for the standard entropy change. This effect appears to be independent of the concentration of the gel matrix to which the cytochrome c is bound, but dependent on the ionic strength of the surrounding solution.     

The capacitance of skeletal muscle fibers in solutions of low ionic strength

The capacitance of skeletal muscle fibers was measured by recording with one microelectrode the voltage produced by a rectangular pulse of current applied with another microelectrode. The ionic strength of the bathing solution was varied by isosmotic replacement of NaCl with sucrose, the [K] [Cl] product being held constant. The capacitance decreased with decreasing ionic strength, reaching a value of some 2 µF/cm² in solutions of 30 mM ionic strength, and not decreasing further in solutions of 15 mM ionic strength. The capacitance of glycerol-treated fibers did not change with ionic strength and was also some 2 µF/cm². It seems likely that lowering the ionic strength reduces the capacitance of the tubular system (defined as the charge stored in the tubular system), and that the 2 µF/cm² which is insensitive to ionic strength is associated with the surface membrane. The tubular system is open to the external solution in low ionic strength solutions since peroxidase is able to diffuse into the lumen of the tubules. Twitches and action potentials were also recorded from fibers in low ionic strength solutions, even though the capacitance of the tubular system was very small in these solutions. This finding can be explained if there is an action potential—like mechanism in the tubular membrane.     

Changes in chromatin folding in solution

The formation of higher order structures by nucleosome oligomers of graded sizes with increasing ionic strength has been studied in solution, by measuring sedimentation coefficients. Nucleosome monomers and dimers show no effect of ionic strength at the concentrations used, while trimers to pentamers show a linear dependence of the logarithm of sedimentation coefficient upon the logarithm of ionic strength between 5 and 25 mm, but no dependence above 25 mm. Between pentamer and hexamer a change occurs and the linear relationship is observed up to ionic strength 125 mm with hexamer and above.The simple power-law dependence of the sedimentation coefficient upon the ionic strength (s ∝ Iⁿ) is observed up to nucleosome 30mers, but by 60mer a jump in the sedimentation coefficient occurs between ionic strengths 45 and 55 mm, with the power-law applying both above and below the jump. Removal of histone H1 and non-histone proteins lowers the overall sedimentation rate and abolishes the jump.Cross-linking large oligomers at ionic strength 65 mm stabilizes the structure in the conformation found above the jump, leading to a simple power-law dependence throughout the range of ionic strength for cross-linked material. Cleavage of the cross-links restores the jump, presumably by allowing the conformational transition that causes it. Large oligomers are indistinguishable in sedimentation behaviour whether extracted from nuclei at low ionic strength or at 65 mm and maintained in the presence of salt.We interpret these results, together with the detailed electron microscopic studies reported by Thoma et al. (1979) under similar salt conditions, as showing the histone H1-dependent formation of superstructures of nucleosomes in solution induced by increasing ionic strength. The unit of higher order structure probably contains five or six nucleosomes, leading to the change in stability with hexamer. Although this size corresponds to the lower limit of size suggested for “superbeads” (Renz et al., 1977), we see no evidence that multiples of six nucleosomes have any special significance as might be predicted if superbeads had any structural importance. Rather, our results are compatible with a continuous pattern of condensation, such as a helix of nucleosomes (see e.g. Finch & Klug, 1976). The jump in sedimentation observed between ionic strengths 45 and 55 mm, together with the effect of cross-linking, suggests the co-operative stabilization of this structure at higher ionic strengths. A plausible hypothesis is that the turns of the solenoid are not tightly bonded in the axial direction below 45 mm, but come apart due to the hydrodynamic shearing forces in the larger particles leading to less compact structures with slower sedimentation rates. Above 55 mm the axial bonding is strong enough to give a stable structure of dimensions compatible with the 30 nm structures observed in the cell nucleus.     

Differential electrostatic stabilization of A-, B-, and Z-forms of DNA

The static accessibility discrete charge algorithm for protein charge interactions is extended to the case of linear polyelectrolytes. In this model, the effective dielectric value between surface charge sites depends predominantly on the solvent ionic strength and the solvent accessibilities of the charge sites. This treatment accounts for the phenomena of specific ion binding in the context of a general electrostatic effect [Matthew and Richards (1982) Biochemistry 21 , 4989]. Specific ion sites are determined by locating areas of high electrostatic potential at the solvent interface of the macromolecule. At a given ionic strength the calculated potential at a site is taken to describe a binding constant and therefore the ion site occupancy. For a 20-base-pair fragment of B-DNA, net charge of ?40, 16 ion sites are indicated in the minor groove. The partial occupancy of each site increases from 0.2 to 0.5 as the ionic strength is increased from 0.01 to 0.50. Over the same range of ionic strength, the electrostatic free energy of this charge array is calculated to change from +0.6 to ?0.05 kcal/bp. Parallel behavior is predicted for A- and Z-DNA charge geometries. The most stable configuration, based on electrostatic criteria, at high ionic strength (I = 0.1–0.5) is that of Z-DNA. In this range, the ratio of “bound” sodium to phosphate is predicted to be less than 0.4.     

Electrostatic effects on the kinetics of bound enzymes.

1. The effect of the interaction between the charged matrix and substrate on the kinetic behaviour of bound enzymes was investigated theoretically. 2. Simple expression is derived for the apparent Km. 3. The apparent Km can only be used for the characterization of the electrostatic effect of the ionic strength does not vary with the substrate concentration. 4. The deviations from Michaelis-Menton kinetics are graphically illustrated for cases when the ionic strength varies with the substrate concentration. 5. The inhibition of the bound enzyme by a charged inhibitor at constant ionic strength is characterized by an apparent Ki. 6. When both the inhibitor concentration and the ionic strength change there is no apparent Ki, and the inhibition profile is graphically illustrated for this case. 7. Under certain conditions the electrostatic effects manifest thenselves in a sigmoidal dependence of the enzyme activity on the concentration of the substrate or inhibitor.