Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.     

Cation dependence of guinea pig epidermal cell spreading

Summary To understand the earliest phases of epidermal cell spreading we have sought a defined in vitro system. We studied the divalent cation dependence of guinea pig epidermal cell spreading in media containing varying concentrations of cations. No spreading occurred in calcium-magnesium-free Dulbecco's modified Eagle's medium (CMF-DME) in the presence of cation-free fetal bovine serum; however, significant spreading occurred if the medium was supplemented with Mg⁺⁺ plus Ca⁺⁺ or Mg⁺⁺ alone. Supplementing with Ca⁺⁺ alone led to much less spreading. These cations in CMF-DME did not support spreading in the absence of serum or the presence of serum albumin. Assaying cell spreading in a simple salt solution consisting of NaCl, KCl, Tris buffer, pH 7.4 plus dialyzed serum and a series of divalent cation supplements (Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Co⁺⁺, Zn⁺⁺, Ni⁺⁺), showed that only Mg⁺⁺ and Mn⁺⁺, and to a lesser extent, Ca⁺⁺, supported cell spreading. In contrast to Mg⁺⁺, however, Mn⁺⁺ could support spreading in the absence of whole serum if serum albumin were present. Although Mn⁺⁺ plus serum albumin supported more rapid spreading at lower cation concentrations than Mg⁺⁺ plus serum, equal concentrations of Ca⁺⁺ completely blocked the Mn⁺⁺ effect. In contrast to the increasing cell spreading, which occurred in Mg⁺⁺-containing medium with time, cell death occurred in Mn⁺⁺-containing medium by 24 h. Consonant with studies from other laboratories, human foreskin fibroblasts spread in Mn⁺⁺-containing salt solution in the absence of protein supplements. These experiments indicate for epidermal cell spreading that Mg⁺⁺ is the important cation in tissue culture media, that under proper cation conditions epidermal cells do not need a specific spreading protein (i.e. a protein that has been demonstrated to support cell spreading), that Mn⁺⁺ and Mg⁺⁺-induced spreading seem to represent different mechanisms, that fibroblastic and epidermal cells have different cation requirements for in vitro spreading, and that the crucial role cations play in cell spreading remains to be elucidated. This work was supported in part by Public Health Service grant CA34470-01 (KSS) awarded by the National Cancer Institute, Bethesda, Md.     

The Role of Polyphosphates in the Transport Mechanism of Glucose in Yeast Cells

Several cations inhibit anaerobic fermentation of glucose by intact yeast cells. Some ions (e.g. Hg⁺⁺) penetrate into the cytoplasm and cause an irreversible inhibition of fermentation. Other ions (e.g. UO₂⁺⁺, Ni⁺⁺, and Co⁺⁺) are reversibly bound to a substance at the outside of the yeast cell identified as polyphosphate. Although the cations are bound to exactly the same extent, their influences on fermentation differ greatly. Thorium ions are bound not only to the polyphosphates, but in addition, to phosphatides in the cell membrane. Under circumstances in which glucose is transported into the cell, the amount of polyphosphate in the outer face of the membrane decreases considerably. If yeast is poisoned with monoiodoacetate, the number of glucose molecules that can still be taken up equals the original number of cation-binding sites at the outer surface of the membrane. These data suggest that one molecule of glucose is taken up in connection with the disappearance of one polyphosphate monomer. The hypothesis is framed that the uptake of glucose into the yeast cell is associated with an enzymic phosphorylation (possibly of the carrier), with polyphosphate as phosphate donor. The inhibition of glucose uptake caused by certain metal ions may be the consequence of induced changes in the spatial arrangement of polyphosphate chains; the greater the change in configuration, the larger is the inhibition.     

The active transport of Mg++ and Mn++ into the yeast cell

Certain bivalent cations, particularly Mg⁺⁺ and Mn⁺⁺, can be absorbed by yeast cells, provided that glucose is available, and that phosphate is also absorbed. The cation absorption is stimulated by potassium in low concentrations, but inhibited by higher concentrations. From the time course studies, it is apparent that the absorption rather than the presence of phosphate and the potassium is the important factor. Competition studies with pairs of cations indicate that binding on the surface of the cell is not a prerequisite to absorption. The absorption mechanism if highly selective for Mg⁺⁺ and Mn⁺⁺, as compared to Ca⁺⁺, Sr⁺⁺, and UO₂⁺⁺, whereas the binding affinity is greatest for UO₂⁺⁺, with little discrimination between Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, and Sr⁺⁺. In contrast to the surface-bound cations which are completely exchangeable, the absorbed cations are not exchangeable. It is concluded that Mg⁺⁺ and Mn⁺⁺ are actively transported into the cell by a mechanism involving a phosphate and a protein constituent.     

EARLY OBSERVATIONS ON SOME LIMNOLOGICAL CHARACTERISTICS OF THREE ORANGE FREE STATE IMPOUNDMENTS

SUMMARY

The first limnological investigations on three impoundments in the Orange Free State are described. No thermal stratification was found and water temperatures below 8,6°C were not measured. The waters were turbid and the unfavourable underwater light climate was the major factor limiting algal growth. The catchment areas influenced the ionic composition of the waters and large quantities of ions were adsorbed onto the suspended silt.     

CHARACTERIZATION OF LYMPHOCYTE TRANSFORMATION INDUCED BY ZINC IONS

Lymphocyte cultures from all normal human adults are stimulated by zinc ions to increase DNA and RNA synthesis and undergo blast transformation. Optimal stimulation occurs at 0.1 mM Zn⁺⁺. Examination of the effects of other divalent cations reveals that 0.01 mM Hg⁺⁺ also stimulates lymphocyte DNA synthesis. Ca⁺⁺ and Mg⁺⁺ do not affect DNA synthesis in this culture system, while Mn⁺⁺, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, and Ni⁺⁺ at concentrations of 10^-7–10^-3 M are inhibitory. DNA and RNA synthesis and blast transformation begin to increase after cultures are incubated for 2–3 days with Zn⁺⁺ and these processes reach a maximum rate after 6 days. The increase in Zn⁺⁺-stimulated lymphocyte DNA synthesis is prevented by rendering cells incapable of DNA-dependent RNA synthesis with actinomycin D or by blocking protein synthesis with cycloheximide or puromycin. Zn⁺⁺-stimulated DNA synthesis is also partially inhibited by 5'-AMP and chloramphenicol. Zn⁺⁺ must be present for the entire 6-day culture period to produce maximum stimulation of DNA synthesis. In contrast to its ability to independently stimulate DNA synthesis, 0.1 mM Zn⁺⁺ inhibits DNA synthesis in phytohemagglutinin-stimulated lymphocytes and L1210 lymphoblasts.     

Mechanisms of adhesion among cells of the early chick blastoderm. Role of calcium ions in the adhesion of extraembryonic endoderm cells

Summary Cells from the extraembryonic endoderm of the gastrulating chick embryo adhere to one another in the absence of divalent cations. The addition of Mg²⁺ ions to the medium has no effect on the aggregation kinetics but the addition of Ca²⁺ ions increases the number of cells which aggregate and also stabilizes adhesion. Some aggregation also occurs when cells are suspended in saline devoid of Ca²⁺ and Mg²⁺ ions and supplemented with EGTA, a Ca²⁺ ion complexing agent, but adhesion is not stabilized. Shear sensitive and shear resistant bonds form in Ca-containing as well as in EGTA-containing saline. These results suggest that extraembryonic endoderm cells have Ca²⁺ indepedent and Ca²⁺ dependent mechanisms of adhesion.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

Effect of magnesium and calcium ions on blocking of synaptic transmission by adenosine in isolated rat hippocampal slices

Dependence of the inhibitory action of adenosine on the extracellular composite EPSP on the concentrations of Mg and Ca cations in the medium was investigated in isolated slices of rat hippocampusin vitro. Extracellular EPSPs were derived in the region of apical dendrites of pyramidal cells in area CA₁ during stimulation of Schaffer's collaterals. The blocking action of bivalent cations (an increase in Mg⁺⁺ or a decrease in Ca⁺⁺) developed almost five times more slowly than the action of adenosine. An increase in the external Mg⁺⁺ concentration potentiated whereas a decrease weakened the inhibitory action of adenosine. Ca⁺⁺ ions had the opposite effect. Antagonistic relations were exhibited between Mg⁺⁺ and Ca⁺⁺ ions. Analysis of dose-response curves for adenosine showed that during a simultaneous increase in the extracellular Ca⁺⁺ and decrease in Mg⁺⁺ concentrations, not only was the maximal effect of adenosine reduced, but so also was its binding constant with the receptor. The results suggest that antagonism between Ca cations and adenosine is mixed in character — both competitive and noncompetitive. The possible mechanism of the inhibitory action of adenosine on synaptic transmission and the role of bivalent cations in this process are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 532–539, July–August, 1984.     

23Na NMR investigation of the interaction between DNA and divalent metallic cations

The aim of this study is to follow the thermodynamic behaviour of Na⁺ ions, acting as natural counterions of DNA, in the presence of divalent metal ions, by using the²³Na NMR technique. With the help of the²³Na entropy of fluctuations concept introduced by Lenk, we propose the following decreasing sequence: Mg⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, and Cu⁺⁺, for the magnitude of divalent metal ions interactions with DNA phosphate sites.     

Further Studies on the Roles of Sodium and Potassium in the Generation of the Electro-Olfactogram : Effects of mono-, di-, and trivalent cations

In the negative EOG-generating process a cation which can substitute for Na⁺ was sought among the monovalent ions, Li⁺, Rb⁺, Cs⁺, NH₄⁺, and TEA⁺, the divalent ions, Mg⁺⁺, Ca⁺⁺, Sr⁺⁺, Ba⁺⁺, Zn⁺⁺, Cd⁺⁺, Mn⁺⁺, Co⁺⁺, and Ni⁺⁺, and the trivalent ions, Al⁺⁺⁺ and Fe⁺⁺⁺. In Ringer solutions in which Na⁺ was replaced by one of these cations the negative EOG's decreased in amplitude and could not maintain the original amplitudes. In K⁺-Ringer solution in which Na⁺ was replaced by K⁺, the negative EOG's reversed their polarity. Recovery of these reversed potentials was examined in modified Ringer solutions in which Na⁺ was replaced by one of the above cations. Complete recovery was found only in the normal Ringer solution. Thus, it was clarified that Na⁺ plays an irreplaceable role in the generation of the negative EOG's. The sieve hypothesis which was valid for the positive EOG-generating membrane or IPSP was not found applicable in any form to the negative EOG-generating membrane. The reversal of the negative EOG's found in K⁺- , Rb⁺- , and Ba⁺⁺-Ringer solutions was attributed to the exit of the internal K⁺. It is, however, not known whether or not Cl^- permeability increases in these Na⁺-free solutions and contributes to the generation of the reversed EOG's.     

Dielectric properties of polyelectrolytes. III. Effect of divalent cations on dielectric increment of polyacids

Dielectric dispersion of polyacrylic acid (PAA) and polystyrene sulfonic acid (PSS) was measured in the presence of divalent cations. Effects of divalent ions were studied by neutralization with varying ratios of sodium hydroxide and divalent base concentration, addition of salts of divalent cations, and neutralization with divalent bases only. Two dispersion regions were observed in all cases, i.e., low-frequency dispersion (10²–10⁴ Hz) and high-frequency dispersion (10⁵–10⁶ Hz). The dielectric increment increases in the presence of sodium and alkaline earth metal ions together, but not with sodium and transition metal ions. This is due to the increment of low-frequency dispersion and is attributable to the fluctuation of bound counterions which is explained by our theory previously reported.¹ In the case of PAA neutralized with large fractions of divalent ions, or with divalent ions only, the increment is very small because of reduction of the fluctuation by interaction between bound ions at the neighboring sites and reduction of the effective length of polyion probably due to chelation by divalent ions. There are some differences among the effects of Mg⁺⁺, Ca⁺⁺, and Ba⁺⁺ on dielectric increment which may result from affinity or chelating ability of these ions.     

QUANTITATIVE STUDIES OF PHAGOCYTOSIS : Kinetic Effects of Cations and Heat-Labile Opsonin

Kinetic analysis of the initial ingestion rate of albumin-coated paraffin oil particles by human granulocytes and rabbit alveolar macrophages was undertaken to study the mechanism of action of cations and of heat-labile opsonin on engulfment. The rate of uptake of the particles was stimulated by Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, or Co⁺⁺. At high concentrations (> 20 mM) Ca⁺⁺ and Mg⁺⁺ inhibited the rate of ingestion. Treatment of the particles with fresh serum (heat-labile opsonin) also stimulated the rate of ingestion. ¹²⁵I-labeled C3 was bound to the particles during opsonization. C3-deficient human serum lacked opsonic activity, which was restored by addition of purified C3. Normal, C2-deficient, and hereditary angioneurotic edema sera had equivalent opsonic activity. The serum opsonic activity thus involved C3 fixation to the particles by means of the properdin system. Although Mg⁺⁺ and heat-labile opsonin both accelerated the maximal rates of ingestion of the particles, neither altered the particle concentrations associated with one-half maximal ingestion rates. Opsonization of the particles markedly diminished the concentrations of divalent cations causing both stimulatory and inhibitory effects on ingestion rates and altered the shapes of the cation activation curves. ⁴⁵Ca was not bound to the particles during opsonization. The results are consistent with a mechanism whereby divalent cations and heat-labile opsonin activate ingestion by stimulating the work of engulfment rather than by merely enhancing cell-particle affinity, and whereby heat-labile opsonin acts by potentiating the effects of divalent cations.     

On the selective adsorption of cations in the cell wall of the green algaValonia utricularis

The selective adsorption of the cations Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ by the cell wall of the Mediterranean algaValonia utricularis (Siphonocladales, Chlorophyceae) from sea water of 40 %. S was investigated by extraction of cell-wall preparations, eluted before in 1.1 mol methanol (adjusted to pH 8) with 0.1 n formic acid in a Soxhlet apparatus. Na⁺ and K⁺ were determined by flame photometry, Mg⁺⁺ and Ca⁺⁺ by complexometric titration with EDTA. From calculation of the dry weight:fresh weight ratios and the chloride determinations in the eluates, the Donnan free-space fraction of the total cell-wall volume was calculated to about 35 %, and the analytical results of the cation concentrations in the extracts expressed asVal cm^–3 DFS. This calculation is based on the assumption that the acidic groups of the noncellulosic matrix material, carrying negative charges by dissociation at the reaction of sea water (ph about 8) are responsible for the adsorption of cations by exhibition of a Donnan effect. The results obtained show clearly that besides the divalent cations Mg⁺⁺ and Ca⁺⁺, which according to the physico-chemical laws of the Donnan distribution must be relatively accumulated to the second power of the monovalent ones, potassium is also enriched by selective adsorption, and the K⁺:Na⁺ ratio increased significantly compared with that in sea water. This seems to indicate that the strength of attraction between the cations and the negative sites is dependent on the radii of the ions and the state of hydration and/or polarisation of the ions and binding sites.     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

Discrimination between Alkali Metal Cations by Yeast : II. Cation interactions in transport

K⁺ is a competitive inhibitor of the uptake of the other alkali metal cations by yeast. Rb⁺ is a competitive inhibitor of K⁺ uptake, but Li⁺, Na⁺, and Cs⁺ act like H⁺. At relatively low concentrations they behave as apparent noncompetitive inhibitors of K⁺ transport, but the inhibition is incomplete. At higher concentrations they inhibit the remaining K⁺ transport competitively. Ca⁺⁺ and Mg⁺⁺ in relatively low concentrations partially inhibit K⁺ transport in an apparently noncompetitive manner although their affinity for the transport site is very low. In each case, in concentrations that produce "noncompetitive" inhibition, very little of the inhibiting cation is transported into the cell. Competitive inhibition is accompanied by appreciable uptake of the inhibiting cation. The apparently noncompetitive effect of other cations is reversed by K⁺ concentrations much higher than those necessary to essentially "saturate" the transport system. A model is proposed which can account for the inhibition kinetics. This model is based on two cation-binding sites for which cations compete, a carrier or transporting site, and a second nontransporting (modifier) site with a different array of affinities for cations. The association of certain cations with the modifier site leads to a reduction in the turnover of the carrier, the degree of reduction depending on the cation bound to the modifier site and on the cation being transported.     

HYDROCHEMISTRY OF THE UPPER ORANGE RIVER CATCHMENT

SUMMARY

A four year pre-impoundment study of the catchment of the Hendrik Verwoerd Dam was conducted. The Orange River was the major source of dissolved chemicals and suspended sediments, while the Caledon River contributed 24 and 30% respectively, and the Kraai River 9 and 8% respectively. The upper reaches of the Orange River catchment contributed mainly to the dissolved chemical and sediment loads, in contrast to the minimal contribution of the lower reaches. This was in spite of the relatively stable geological formations in the upper reaches and the erodable formations in the lower reaches. Dry-wet seasonal variations occurred in nutrient and sediment concentrations and loads, except for the Bell River where constant values were measured. The water was characterized by magnesium and bicarbonate dominance. Adsorption by the suspended sediments was dominated by calcium (potassium in the Bell River) and zinc among the trace elements. Due to its low salinity and sodium adsorption ratio, the water was suitable for irrigation. High magnesium levels made it, however, less attractive for irrigational purposes.

The phosphorus loading rate at the inflow of the Hendrik Verwoerd Dam was only 0,55 g P m^?2 a^?1, which was well below the 1,39 g P m^?2 a^?1 critical rate for eutrophic conditions, applying the Vollenweider Model. Therefore, this impoundment was classified as oligo-mesotrophic.     

Polarographic studies of membrane particles containing Na−K ATPase

Summary The properties of a suspension of membrane particles containing Na–K ATPase have been investigated with the aid of d–c and a–c polarography. In particular, we have studied the interaction of three cations, two very effective enzyme inhibitors and one activator, with the enzyme preparation. Ag⁺ and Cu⁺⁺, which inhibit the enzyme at very low concentrations, bind very strongly. No binding could be found with the activating ion, Tl⁺, however. Adsorption of a substance with an isoelectric point between pH 4 and pH 5.5 occurred at the electrode surface between –0.1 and –1.2 V at pH 7, and was associated with the random currents that appear during the measurements. The random currents arise when the membrane particles collide with the electrode and cause changes in the structure of the electrical double layer. (Added substances that adsorb more strongly at the mercury/water interface eliminate the random currents.) The adsorbed film impedes the flow of the free Ag⁺ and Cu⁺⁺ ions, and to a smaller extent, the flow of Tl⁺ ions. The differences between the binding of inhibiting and activating ions are correlated with their effects on the ATPase enzyme activity.     

Characteristics of lead adsorption byUndaria pinnatifida

Summary We reportUndaria pinnatifida as a potential biosorbent for lead removal.U. pinnatifida exhibited approximately 350 mg of lead uptake capacity per gram of dry biomass within a pII range of 3 to 4. The major cellular component ofU. pinnatifida in lead binding was thought as alginic acid existing in the cell wall.U. pinnatifida adsorbed lead ion selectively over other alkaline metal ions such as K⁺, Na⁺, Mg⁺⁺ and Ca⁺⁺. It was confirmed by using instrumental analysis including EDX and XPS thatU. pinnatifida adsorbed lead ions on the surface of itself.This research was supported by Bioprocess Engineering Research Center and Kyunggi Chemical Industrial Company.     

Triple helix formation and disulphide bonding during the biosynthesis of glomerular basement membrane collagen.

White erythrocyte membranes, or ghosts, were monoconcave discocytes when incubated in 50mM N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid titrated to pH 7.4 with triethanolamine. If 3mM MgCl₂ was included in the incubation medium, the ghosts were predominantly echinocytes. The echinocytic form could also be induced by Co⁺⁺, Ni⁺⁺, Li⁺, Na⁺, K⁺, $\text{NH}{}_4{}^{\mathrm{+}}$ and tetramethylammonium ion, all as chloride salts. The concentration of cation necessary for 50% of the ghosts to be echinocytes was correlated with the hydrated charge density of the cation with the most highly charged cations being the most effective. The cations Ca⁺⁺, Sr⁺⁺, Ba⁺⁺ and La⁺⁺⁺, (also as chloride salts) did not induce the normal echinocytic form, but at high levels induced a few misshapen forms with some resemblance to echinocytes. Instead Ca⁺⁺, Sr⁺⁺, Ba⁺⁺ and La⁺⁺⁺ suppressed the formation of echinocytes in the presence of Mg⁺⁺ and other ions. This suggests the presence of a specific Ca⁺⁺ binding site important to shape control in the erythrocyte membrane.