Studies on isolated cell components. IV. The effect of various solutions on the isolated rat liver nucleus

1. The effects of morganic ions, electrolyte concentration, and pH on the appearance and volume of the isolated rat liver nucleus have been studied. Nuclei were isolated by differential centrifugation in a buffered salt-sucrose mixture at pH 7.1. Nuclear volumes were determined photographically. 2. In solutions of NaCl, of KCl, and in potassium phosphate buffers the nuclear volume decreased markedly with an increase in concentration from 0.001 M to 0.05 M but remained essentially constant with further increase in concentration to 1.0 M. The effects of CaCl(2) and MgCl(2) differed from those of NaCl and KCl in that a smaller volume was obtained in concentrations less than 0.15 M, and in the case of CaCl(2) an increase in volume was obtained in more concentrated solutions. The volume changes are considered to be due primarily to ionic effects on the nuclear colloids rather than to osmotic behavior. 3. Treatment of nuclei with DNAase prevented the characteristic volume changes resulting from ion effects, suggesting the importance of DNA in nuclear volume changes. 4. The optical changes in isolated nuclei in various concentrations of KCl, NaCl, CaCl(2), MgCl(2), and in potassium phosphate buffers as observed under phase contrast illumination are described. CaCl(2) gave the most marked nuclear changes from the conditions in the uninjured cell and caused shrinkage and granulation in 0.001 M concentration. The effects of CaCl(2) were also manifested in 0.88 M sucrose, in mixtures with monovalent salts, and in serum. Changes in nuclear volume and optical appearance which occurred in salt solutions and in 0.1 N HCl were readily reversible. 5. Nuclear volume remained constant between pH 8.91 and 5.12 and decreased in more acid solutions. 6. Sucrose had no appreciable osmotic effect, and in hyperosmotic solution. (0.88 M) nuclei showed swelling and rupture comparable to that in distilled water. 7. The results are considered in relation to the requirements of nuclear isolation media. 8. Rat liver nuclei isolated in a buffered salt-sucrose medium by differential centrifugation exhibited a pattern of size distribution similar to that of fixed nuclei but were of considerably larger volume. The ratio of the volumes of the peak frequencies of the two chief size groups was 1:1.9.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

Competitive substitution of hexammine cobalt(III) for Na+ and K+ ions in oriented DNA fibers

Competition of the trivalent cation, Co(NH3)(3+)(6), with K+ and Na+ ions in binding to DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH), containing different combinations and concentrations of KCl and NaCl and constant concentration (0.8 mM) of Co(NH3)(6)Cl(3). The degree of Co(NH3)(3+)(6) binding to DNA does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na+ or K+). The ion exchange selectivity coefficient of monovalent-trivalent ion competition, D(1)(c3), increases with the concentration of Me+, C(o)(+), and the monotonic dependence of log D(1)(c3) vs log C(o)(+) has an inflection between 100 and 300 mM that is caused by a structural transformation of DNA from A- to B-form. The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, discretely charged polyions with density and spatial distribution of the charged groups modeling B- and A-forms of DNA. The GCMC method for discretely charged models of the DNA polyion produces a quantitative agreement with experimental data on trivalent-monovalent ion competition in dependence on DNA structural state and salt concentration. Based on this and previous studies it is concluded that the affinity of DNA for the cations decreases in the order Co(NH3)(3+)(6) > Ca2+ > Mg2+ > Na+ approximately K+ > Li+. DNA does not exhibit selectivity for Na+ or K+ in ethanol/water solutions either in the absence or in the presence of Co(NH3)(3+)(6), Ca2+, and Mg2+.     

盐度对木榄幼苗某些金属元素累积的影响及钙的效应

通过对 40 0mmol·L^-1NaCl及不同浓度CaCl² 溶液处理的沙基培养的红树植物木榄幼苗各器官的K、Ca、Na、Mg含量的测定分析,结果表明,木榄幼苗在 40 0mmol·L^-1NaCl培养时各器官中积累大量的Na,造成K/Na和Ca/Na比值降低,幼苗干物质积累下降.补充Ca能减少Na在幼苗体内的累积,提高K/Na和Ca/Na,增加干物质积累,从而缓解盐胁迫,10～15mmol·L^-1CaCl² 为适宜浓度.本文的结论是在高盐度培养时木榄幼苗能够吸收大量的无机离子,增强渗透调节能力,保持对K/Na的高选择吸收性.这些特性是木榄对河口、海岸高盐度生境适应的主要生理机制,而生境土壤中适宜的Ca/Na也是其能够生存繁衍的重要原因之一.     

The osmotic pressure of chondroitin sulphate solutions: experimental measurements and theoretical analysis

We used equilibrium dialysis to measure the osmotic pressure of chondroitin sulphate (CS) solutions as a function of their concentration and fixed charge density (FCD) and the ionic strength and composition of the solution. Osmotic pressure varied nonlinearly with the concentration of chondroitin sulphate and in 0.15 M NaCl at FCDs typical of uncompressed cartilage (approximately 0.4 mmol/g extrafibrillar H2O) was approximately 3 atmospheres. Osmotic pressure fell by 60% as solution ionic strength increased up to about 1 M, but remained relatively constant at higher ionic strengths. The ratio of Ca2+ to Na+ in the medium was a minor determinant of osmotic pressure. The data are compared with a theoretical model of the electrostatic contribution to osmotic pressure calculated from the Poisson-Boltzmann equation using a rod-in-cell model for CS. The effective radius of the polyelectrolyte rod is taken as a free parameter. The model qualitatively reproduces the non-linear concentration dependence, but underestimates the osmotic pressure by an amount that is independent of ionic strength. This difference, presumably arising from oncotic and entropic effects, is approximately 1/3 of the total osmotic pressure at physiological polymer concentrations and ionic strength.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

Mechanism of CaCl2-induced actin polymerization

The CaCl2 concentration dependence of the rate of actin filament elongation and of the actin monomer concentration at steady state with actin polymer (the critical actin concentration) has been investigated. A relative rate of actin filament elongation from actin polymer intermolecularly cross-linked with N,N'-p-phenylenebis(maleimide) showed a sigmoidal dependence on the concentration of CaCl2 used to induce actin polymerization. This result is shown to be consistent with a model in which only actin monomer containing five equivalently bound Ca2+ ions (Ka = 2 mM-1) is capable of addition to actin polymer. A relative dissociation rate constant for actin monomer removal from polymer was calculated from the product of the critical actin concentration and the relative elongation rate constant and was found to be virtually independent of CaCl2 concentration. The relationship between Ca2+ binding sites on actin and the CaCl2 concentration dependence of the kinetics of actin filament elongation is discussed.     

Effect of cationic size on gelation temperature and properties of gelatin hydrogels

Effect of Na+, K+ and Ca2+ on gel transition temperature (Tg) of gelatin hydrogels (5%, w/v) has been studied by oscillatory rheology in the salt concentration range I = 0.01-0.1 M, which showed increase in Tg with salt concentration with the trend for Tg showing Ca2+ > K+ > Na+. The dynamic light scattering (DLS) measurements in the sol state (T>Tg) showed two distinct relaxation modes whereas only a gel mode was observed in the gel state in all the samples which contained significant amount of heterodyne contribution. Low frequency (1.5 rad/s) isochronal storage modulus data revealed the formation of strong gel in presence of CaCl2 compared to that of NaCl and KCl situations. The slow mode relaxation and heterodyne parameter obtained from DLS data indicate the presence of larger clusters in Ca2+ gels.     

盐胁迫下盐地碱蓬体内无机离子含量分布特点的研究

用不同浓度NaCl溶液处理盐地碱蓬（Suaeda salsa)植株后,测定并比较老叶、幼叶及根部的无机离子含量和对K的选择性,叶片及根部的Na^ 、Cl^-含量随盐度的增加而升高,且累积趋势相似,盐胁迫下根部Na^ 、Cl^-及总离子含量（K^ 、Na^ 、Ca^2 ,NO3^-,Cl^-)明显低于叶片,说明盐地碱蓬地盐胁迫下,以叶片优先积累大量离子（如Na^ ,Cl^-) 为其适应特征。NaCl处理下,叶片的K^ ,Ca^2 含量低于对照,但随盐度的增加保持相对稳定,而根部K^ 含量,K/Na比、对K的选择性则高于叶片,这对盐胁迫下地上部的K^ 亏缺有一定补偿作用。低盐度处理（100mmol/LNaCl)促进NO3^-的吸收,另外随盐度的增加,叶片渗透势下降,渗透调节能力增强,幼叶渗透势低于老叶,但渗透调节能力相同。     

A neutron diffraction study of the influence of ions on phospholipid membrane interactions.

Neutron diffraction is used to examine the effects of Ca2+ and ClO4- ions on interactions and some structural features of dipalmitoylphosphatidylcholine membranes in both solid and fluid lamellar phases. The results are described within the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with reference to electrostatic, van der Waals, and hydration components of disjoining pressure. The Hamaker constants are evaluated under equilibrium conditions. Addition of 100 mM CaCl2 to the aqueous phase substantially increases the lamellar repeat spacing (d), which is interpreted in terms of adsorption of Ca2+ ions to bilayers followed by electrostatic repulsion between membranes. The rise of NaClO4 concentration in the presence of 100 mM CaCl2 leads to gradual decrease in d, evidently resulted from the diminution of Ca(2+)-induced positive surface potential by both electrostatic screening and binding of ClO4- ions. In the absence of CaCl2, elevation of NaClO4 concentration to 100-300 mM drastically enhances the repeat spacing and then dramatically decreases d at about 1 M NaClO4. Estimation of the hydration coefficients showed that the pronounced decrease of the repeat spacing at high NaClO4 concentrations was resulted mainly from the (partial) disruption of the structure of intermembrane bound water by chaotropic ClO4- ions and subsequent decrease in hydration repulsive pressure. Moreover, in the case of solid membranes (20 degrees C) high concentrations of ClO4- induced formation of interdigitated phase paralleled with marked reduction in bilayer thickness and corresponding increase in the effective cross-sectional area per lipid molecule.     

Interaction of DNA with divalent metal ions

The interaction between the native DNA macromolecules and Ca2+, Mn2+, Cu2+ ions in solutions of low ionic strength (10(-3) M Na+) is studied using the methods of differential UV spectroscopy and CD spectroscopy. It is shown that the transition metal ions Mn2+ exercise binding to the nitrogen bases of DNA at concentrations approximately 5 x 10(-6) M and form chelates with guanine of N7-Me(2+)-O6 type. Only at high concentrations in solution (5 x 10(-3) M) do Ca2+ ions interact with the nitrogen bases of native DNA. In the process of binding to Ca2+ and Mn2+ the DNA conformation experiences some changes under which the secondary structure of the biopolymer is within the B-form family. The DNA transition to the new conformation is revealed by its binding to Cu2+ ions.     

Interaction and conformational changes of chromatin with divalent ions.

We have investigated the interaction of divalent ions with chromatin towards a closer understanding of the role of metal ions in the cell nucleus. The first row transition metal ion chlorides MnCl2, CoCl2, NiCl2 and CuCl2 lead to precipitation of chicken erythrocyte chromatin at a significantly lower concentration than the alkali earth metal chlorides MgCl2, CaCl2 and BaCl2. A similar distinction can be made for the compaction of chromatin to the "30 nm" solenoid higher order structure which occurs at lower MeCl2 concentration in the first group but at the same MeCl2 concentration within each group. In other experiments in which mixed solutions of NaCl and of MgCl2 were examined, it is shown that increasing NaCl concentration leads to increasing solubility in the presence of MgCl2. Best compaction of chromatin was obtained at 40 mM NaCl and 0.8 mM MgCl2 at a value A260 approximately 0.8. Similar experiments were undertaken with mixtures of NaCl and MnCl2.     

Change in the activity of calcium ions during illumination of a suspension of visual cell outer segment fragments]

Changes in the activity of calcium ions in the medium containing outer fragments suspension of bovine eye retina rods have been studied by the method of calcium-selective electrodes. Illumination of the suspension increases calcium ion activity in the incubation medium. Photoinduced yield of calcium ions depends on Ca+2 concentration: it equals 0.11+/-0.015 M Ca2+/1m rodopsin in the medium containing 0.1 mM CaCl2 and 0.046+/-0.002Ca2+/1M rodopsin in the medium containing 0.05 mM CaCl2. In the medium containing more than 10(-4) M CaCl2 both an increase and a decrease of Ca2+ ions have been observed.     

Nematocysts (stinging capsules of Cnidaria) as Donnan-potential-dominated osmotic systems

Isolated nematocysts (capsules of stinging cells in Cnidaria) from the freshwater polyp Hydra vulgaris [Weber, J., Klug, M. & Tardent, P. (1987) Comp. Biochem. Physiol. 88B, 855-862] are under a constant internal osmotic pressure of 12.5 MPa. The capsular wall which withstands this considerable pressure has an average elasticity modulus of approximately 1 GPa, enabling the cyst to swell from its relaxed state to more than double its volume. If the large concentrations of Mg2+ and Ca2+ within isolated nematocysts are substituted by alkali ions, the capsular volume increases by 15% and the final osmotic pressure rises to 15.3 MPa whereas after substitution by Ca2+ a decrease to 90% of the volume and 6.5 MPa is observed. Evidence obtained from the osmotic behavior of nematocysts, as well as data from in vitro exchange of their cations, are consistent with a physicochemical model in which the internal osmotic pressure of nematocysts and thus their ability to discharge is governed by the cationic composition of the content and the salt concentrations in the environment.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

Influence of the NaCl or CaCl2 concentration on the structure of heat-set bovine serum albumin gels at pH 7

The structure of heat-set systems of the globular protein bovine serum albumin (BSA) was investigated at pH 7 in different salt conditions (NaCl or CaCl(2)) using light scattering. Cross-correlation dynamic light scattering was used to correct for multiple scattering from turbid samples. After heat treatment, aggregates are formed whose size increases as the protein concentration increases. Beyond a critical concentration that decreases with increasing salt concentration, gels are formed. The heterogeneity and the reduced turbidity of the gels were found to increase with increasing salt concentration and to decrease with increasing protein concentration. The structure of the gels is determined by the strength of the repulsive electrostatic interactions between the aggregated proteins. The results obtained in NaCl are similar to those reported in previous studies for other globular proteins. CaCl(2) was found to be much more efficient in reducing electrostatic interactions than NaCl at the same ionic strength.     

Electrical characteristics in an excitable element of lipid membrane.

Electrical characteristics in a membrane constructed from a porous filter adsorbed with a lipid analogue, dioleoyl phosphate (DOPH), were investigated in a situation interposed between 100 mM NaCl + 3 mM CaCl2 and 100 mM KCl. Calcium ions affected significantly the membrane characteristics. The membrane potential was negative on the KCl side, which implies the higher permeability to K+ than Na+; this tendency was increased by a tiny amount of Ca2+. While the membrane showed a low electrical resistance of several k omega . cm2 under K+/Na+ gradient, it showed several M omega . cm2 by Ca2+. The surface structure of the membrane exhibited many voids in the low-resistance state, but the surface was covered by oil droplets in the high-resistance state. Oscillations of the membrane potential appeared spontaneously with application of the electrical current from the KCl side to the NaCl + CaCl2 side. The frequency was increased with the electrical current. All these results were explained comprehensively using an electrochemical kinetic model taking account of the Ca2+ binding effect, where DOPH assemblies make a phase transition between oil droplets due to Ca2+ and multi-bilayers with excess K+. The oscillation arises from coupling of the phase transition to accumulation and release of K+ or Ca2+. This membrane can be used as an excitable element regulated by Ca2+ in neuro-computer devices.     

The effect of temperature on DNA structural transitions under the action of Cu2+ and Ca2+ ions in aqueous solutions

The work examines the structural transitions of DNA under the action of Cu2+ and Ca2+ ions in aqueous solution at temperatures of 29 and 45 degrees C by ir spectroscopy. Upon binding to the divalent ions studied, DNA transits into the compact state both at 29 and 45 degrees C. In the compact state DNA remains in B-form limits. The compaction process is of high positive cooperativity. As temperature increases the divalent metal ion concentration required to induce DNA compaction decreases in the case of Cu(2+)-induced compaction and increases in the case of Ca(2+)-induced compaction. It is suggested that the mechanism of the temperature effect on DNA compaction in the presence of Cu2+ ions possessing higher affinity for DNA bases differs from that of the temperature influence on Ca(2+)-induced DNA compaction. In the case of copper ions the determining factor is the increase of binding constants of the Cu2+ ions interacting with the denatured parts formed on DNA while in the case of calcium ions it is the decreased screening action of counterions upon the increase of their hydration with temperature. The efficiency of divalent metal ions studied in inducing DNA compaction depends on hydration of counterions. DNA compaction occurs in a narrow interval of Cu2+ concentrations. As the Cu2+ ion concentration increases, DNA compaction is replaced with Cu(2+)-induced DNA aggregation. At elevated temperatures Cu(2+)-induced DNA compaction could acquire a phase transition character.     

Effect of ethanol on structural transitions of DNA and polyphosphates under Ca2+ ions action in mixed solutions

In the present work using the IR spectroscopy method the effect of ethanol on structural transitions of DNA and polyphosphates under the action of Ca2+ ions in mixed solutions containing ethanol (0-25 vol.%) was studied. It was shown that, on its interaction with Ca2+ ions, in aqueous and mixed solutions DNA becomes transformed into compact form. With the increase of concentration of ethanol the degree of Ca2+-induced DNA compactisation rises. It was found that, in mixed solutions containing ethanol, Ca2+-induced DNA compactisation depends not only on the solution's dielectric permeability but also on the solution structure. On stabilisation of the water structure in the presence of low ethanol concentrations a stabilisation of the DNA macromolecule occurs that leads to the increase of the Ca2+ ion concentration necessary for DNA compactisation. Comparison of the effects of ethanol on Ca2+-induced structural transitions in DNA and polyphosphates in mixed solvents permits to suppose that at alcohol concentrations in solution resulting in disruption of the water spatial structure, some peculiarities are observed in the behavior of those molecules whose hydrophobic interactions are essential.     

The influx of calcium ions into human erythrocytes during cold storage

1. When human erythrocytes, suspended in iso-osmotic sucrose containing CaCl(2), are stored at 3 degrees C, Ca(2+) influx into the cells occurs. Simultaneously, efflux of K(+), Na(+), Cl(-) and water takes place and cell volume diminishes. 2. The extent of Ca(2+) influx increases with duration of cold storage and with increasing concentration of Ca(2+) in the suspending medium. 3. Erythrocytes that have been thus loaded with Ca(2+) exhibit Ca(2+) efflux against a concentration gradient when subsequently incubated at 37 degrees C. 4. Ca(2+) influx likewise occurs when the sucrose of the medium is replaced by iso-osmotic solutions of other non-ionized compounds. 5. Replacement of sucrose by iso-osmotic KCl or NaCl greatly diminishes the rate of Ca(2+) influx during cold storage; however, in iso-osmotic choline chloride, Ca(2+) influx is as rapid as in sucrose. 6. Preincubation of erythrocytes in iso-osmotic sucrose at 37 degrees C causes rapid efflux of K(+) and Na(+) and renders the cell membranes highly permeable to Ca(2+) during subsequent cold storage. 7. Preincubation of erythrocytes in iso-osmotic NaCl at 37 degrees C with trypsin or neuraminidase is without effect on the permeability of the membrane towards Ca(2+). 8. The experimental results lead to the conclusion that the main prerequisite for Ca(2+) influx into erythrocytes is the partial depletion of the cells of their univalent cations.