Cation-induced aggregation and fusion of N-acyl-N-methyl-phosphatidylethanolamine vesicles.

Aggregation and fusion of unilamellar vesicles consisting of N-acyl-N-methylphosphatidylethanolamine were studied as a function of mono- and divalent cation concentrations. The aggregation reactions were irreversible processes, as demonstrated by changes in monovalent ion concentrations and by the addition of ethylenediaminetetraacetic acid (EDTA) to chelate divalent cations, suggesting the possibility of some cation-induced vesicle fusion. An increase in the NaCl ionic strength of the vesicle suspension solutions diminishes the threshold concentration for Li+ and K+ and increases that corresponding to Mn2+, Mg2+ and Ca2+. However NaCl concentrations above 300 mM yield smaller threshold values for the divalent cation-induced processes, probably due to the increased size of phospholipid vesicles as the ionic strength of the medium increases.     

Divalent cation-induced aggregation of chromaffin granule membranes.

Divalent cations induce the aggregation of chromaffin granule ghosts (CG membranes) at millimolar concentrations. Monovalent cations produce the same effect at 100-fold higher concentrations. The kinetics of the dimerization phase were followed by light-scattering changes observed in stopped-flow rapid mixing experiments. The rate constant for Ca2+-induced dimerization (kapp) is 0.86-1.0 x 10(9) M-1sec-1, based on the "molar" vesicle concentration. This value is close to the values predicted by theory for the case of diffusion-controlled reaction (7.02 x 10(9) M-1sec-1), indicating that there is no energy barrier to dimerization. Arrhenius plots between 10 degrees and 42 degrees C support this; the activation energy observed, +4.4 Kcal, is close to the value (4.6-4.8 Kcal) predicted for diffusion control according to theory. Artificial vesicles prepared from CG lipids were also found to have cation-induced aggregation, but the rates (values of kapp) were less than 1/100 as large as those with native CG membranes. Also, significant differences were found with respect to cation specificity. It is concluded that the slow rates are due to the low probability that the segments of membrane which approach will be matched in polar head group composition and disposition. Thus large numbers of approaches are necessary before matched segments come into aposition. The salient features of the chromaffin granule membrane aggregation mechanism are as follows: (a) In the absence of cations capable of shielding and binding, the membranes are held apart by electrostatic repulsion of their negatively charged surfaces. (b) The divalent and monovalent cation effects on aggregation are due to their ability to shield these charges, allowing a closer approach of the membrane surfaces. (c) The major determinants of the aggregation rates of CG membranes are proteins which protrude from the (phospholipid) surface of the membrane and serve as points of primary contact. Transmembrane contact between these proteins does not require full neutralization of the surface charge and surface potential arising from the negatively charged phospholipids. (d) After contact between proteins is established, the interaction between membranes can be strengthened through transmembrane hydrogen bonding of phosphatidyl ethanolamine polar head groups, divalent cation-mediated salt bridging, and segregation of phosphatidylcholine out of the region of contact.     

Role of the polar head group stereoconfiguration in the cation-induced aggregation of dimyristoylphosphatidylglycerol vesicles

Cation-induced aggregation of small unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG), the corresponding 3' stereoisomer (3'-DMPG), and their 1:1 mixture was studied as a function of the concentration of different mono- and divalent cations. The order of efficiency, Na+ greater than Li+ greater than K+ greater than Cs+, of the monovalent cations to induce the aggregation of DMPG vesicles is the same for both stereoisomers and their mixture. However, significant differences in the Na+-induced aggregation of 1'-DMPG and 3'-DMPG were evident. The threshold concentration of aggregation by Na+ was 0.35 M for 3'-DMPG, 0.55 M for 1'-DMPG, and 0.50 M for the mixed liposomes. Such difference in the aggregation of DMPG stereoisomers was not observed for the other mono- and divalent cations. The higher affinity of 3'-DMPG for Na+ is suggested to be due to a slightly different favored conformation of the head group glycerol moiety. Aggregation of the stereoisomers by 1 M NaCl was identical, indicating that the differences in the affinity of 1'-DMPG and 3'-DMPG for sodium can be overcome by very high ionic strength. Inclusion of 20 mol % cholesterol in vesicles enhanced the aggregation of 1'-DMPG and decreased the aggregation of 3'-DMPG by Na+ and thus abolished the difference between the two stereoisomers.     

Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH,cations and phosphate

The uptake of Ca²⁺ and Sr²⁺ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca²⁺ and Sr²⁺ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca²⁺ and Sr²⁺ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca²⁺ and Sr²⁺ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Binding of monovalent cations to phosphatidylserine and modulation of Ca2+- and Mg2+-induced vesicle fusion

The effect of several monovalent cations on the Ca2+-induced aggregation and fusion of sonicated phosphatidylserine (PS) vesicles is studied by monitoring the mixing of internal compartments of the fusing vesicles using the Tb/dipicolinic acid assay. The dissociation of the fluorescent Tb-dipicolinate complex which accompanies Ca2+-induced vesicle fusion is determined directly and is due to leakage of contents and entry of medium into vesicles. PS vesicles do not fuse when the medium contains only monovalent cations (at pH 7.4), regardless of the cation concentration or whether there is aggregation of the vesicles. A mass-action kinetic analysis of the data provides estimates for the rate of aggregation, C11, and for the rate of fusion per se, f11. Values of f11 increase dramatically with reduction in monovalent cation concentration and are primarily determined by binding ratios of Ca2+ or Mg2+ per PS. With 300 mM of monovalent cations, the fusion per se is essentially rate-limiting to the overall fusion process and values of f11 are significantly larger with the monovalent cations which bind the least, i.e., according to the sequence tetramethylammonium greater than K+ greater than Na+ greater than Li+. With monovalent cations in concentrations of 100 mM or less, the aggregation is rate-limiting to the fusion and the overall initial fusion rates are determined by an interplay between aggregation and fusion rates. Under conditions of fast aggregation, the Ca2+-induced fusion of small PS vesicles can occur within milliseconds or less.     

Effect of Monovalent Cations on the Aggregation of Tobacco Mosaic Virus by Chondroitin Sulfate

The mechanism of precipitation of tobacco mosaic virus by chondroitin sulfate in the presence of various monovalent cations was investigated kinetically by means of turbidimetry. The virus solution became turbid on the addition of chondroitin sulfate, and led to separation of the virus as a crystalline phase. In the presence of monovalent cations the degree of precipitation of the virus by chondroitin was reduced with the increase in monovalent cation concentration. The order of the reduction was Li⁺<Na⁺<K⁺, which is reversely analogous to the lyotropic series. The least hydrated cation, K⁺, reduced the degree of precipitation of tobacco mosaic virus the most because the radius of the sphere equivalent to chondroitin sulfate was diminished most strongly by K⁺ ion. The effect of the monovalent cations on the precipitation supports our assumption that the virus precipitation results from incompatibility and mutual spatial exclusion between the virus and chondroitin. The maximum turbidities, the initial slopes, and the aggregation half-time were measured by varying the monovalent cation concentrations. The former two parameters decreased with the increase in cation concentration, whereas the aggregation half-time increased. A lag time was present and the effect of the cations increased in the order K⁺<Na⁺<Li⁺.     

Effects of various ionic media on extraction of soluble nuclear proteins and on nuclear ultrastructure

Isolated liver nuclei were extracted 3 times at pH 7.2 with solutions containing either (1) monovalent cations, (2) both mono- and divalent cations, or (3) sucrose solutions containing only divalent cations. The extracted proteins were analysed by two-dimensional acrylamide gel electrophoresis and the ultrastructural alterations of the treated nuclei were examined by electron microscopy. The solutions containing Na⁺ or K⁺ monovalent and Ca²⁺ and Mg²⁺ divalent ions extracted the same amount (18–22 %) of the nuclear proteins. The two-dimensional gel electrophoretic patterns of these extracts were nearly identical and the structures of the nuclear components were well preserved even after 3 times repeated extractions. The solution containing only Na⁺ extracted less protein (14–15 %) than the solutions containing both mono- and divalent cations. Extraction with isotonic NaCl solution altered the nuclear and nucleolar morphology; unlike the other solutions employed, this solution extracted some DNA and histones. The isotonic sucrose solution containing only divalent cations extracted less protein than the other solutions (9–11 %) and produced marked condensation of the chromatin. These analytical and electron microscopic studies showed that mono- and divalent cations play a role in structural organization of chromatin.     

Structures of the mono- and divalent metal nucleotide complexes in the myosin ATPase

The structure of both the mono- and the divalent metal nucleotide complexes active in the myosin subfragment 1 ATPase has been determined using the phosphorothioate analogs of ATP in the presence of various cations. Both the Sp and the Rp diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) were substrates in the presence of Mg2+, Ca2+, Mn2+, Co2+, Zn2+, and Cd2+ as well as with NH4+ and T1+. The Sp/Rp activity ratios obtained were largely independent of the cation. The simplest explanation of these results is that both mono- and divalent cations do not coordinate to the alpha-phosphate group. With adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), essentially only the Sp diastereomer was active with Mg2+ with Sp/Rp ratio of greater 3000. As the divalent metal ion was varied in the series given above, this ratio was progressively lowered to the value of 0.2 found with Cd2+. Similar changes in stereoselectivity were seen with monovalent cations. Thus, with NH4+, an Sp/Rp ratio of 8 was observed, whereas with T1+, this figure was reduced to 0.04. These data indicate that both mono- and divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrate. These results obtained with ATP alpha S and ATP beta S suggest that myosin uses the mono- or divalent cation delta, beta, gamma-bidentate nucleotide chelate as substrate.     

Rapid purification of commercial gellan gum to highly soluble and gellable monovalent cation salts

Commercial gellan gum contains divalent cation contaminants (mainly Ca²⁺ and Mg²⁺) in levels sufficient to neutralize over one-third of its car☐yl groups. Consequently, in order to dissolve gellan gum in water, the mixtures must be heated to more than 90°C. This has prevented applications of gellan gum to such uses as the immobilization of viable enzymes and cells in beads. A rapid two-step method is described here to purify commercial gellan gum to the monovalent cation salts in an overall yield of 85%, through the intermediate free acid form. The gellan monovalent cation salts were highly soluble at temperatures as low as 5°C, and readily gelled upon exposure to solutions of divalent cations. Laboratory-scale preparations of 100 g of gellan monovalent cation salts were readily achieved in a day.     

Potentiation of TRPM7 inward currents by protons

TRPM7 is unique in being both an ion channel and a protein kinase. It conducts a large outward current at +100 mV but a small inward current at voltages ranging from -100 to -40 mV under physiological ionic conditions. Here we show that the small inward current of TRPM7 was dramatically enhanced by a decrease in extracellular pH, with an approximately 10-fold increase at pH 4.0 and 1-2-fold increase at pH 6.0. Several lines of evidence suggest that protons enhance TRPM7 inward currents by competing with Ca(2+) and Mg(2+) for binding sites, thereby releasing blockade of divalent cations on inward monovalent currents. First, extracellular protons significantly increased monovalent cation permeability. Second, higher proton concentrations were required to induce 50% of maximal increase in TRPM7 currents when the external Ca(2+) and Mg(2+) concentrations were increased. Third, the apparent affinity for Ca(2+) and Mg(2+) was significantly diminished at elevated external H(+) concentrations. Fourth, the anomalous-mole fraction behavior of H(+) permeation further suggests that protons compete with divalent cations for binding sites in the TRPM7 pore. Taken together, it appears that at physiological pH (7.4), Ca(2+) and Mg(2+) bind to TRPM7 and inhibit the monovalent cationic currents; whereas at high H(+) concentrations, the affinity of TRPM7 for Ca(2+) and Mg(2+) is decreased, thereby allowing monovalent cations to pass through TRPM7. Furthermore, we showed that the endogenous TRPM7-like current, which is known as Mg(2+)-inhibitable cation current (MIC) or Mg nucleotide-regulated metal ion current (MagNuM) in rat basophilic leukemia (RBL) cells was also significantly potentiated by acidic pH, suggesting that MIC/MagNuM is encoded by TRPM7. The pH sensitivity represents a novel feature of TRPM7 and implies that TRPM7 may play a role under acidic pathological conditions.     

Energized cation transport by complex III (ubiquinone-cytochrome C reductase)

Energized cyclical and net transport of cations and anions has been demonstrated in liposomes containing Complex III using a protamine-aggregation technique. Energization with reduced ubiquinone induces a rapid ion uptake followed by a more gradual efflux upon exhaustion of the available reductant. Both monovalent and divalent cations are transported, but at relatively high concentrations divalent cations are transported in preference to monovalent cations. Results are consistent with a cation:electron ratio of unity.     

Discrimination between monovalent and divalent cations by hydrophobic solvent-saturated membranes containing fixed negative charges

Summary Cellulose acetate-nitrate filters were saturated with hydrophobic solvent and interposed between various aqueous solutions. The membranes thus formed are cation permselective. The discrimination between a monovalent cation such as K⁺ and the alkaline earth group divalent cations is very sharp. The discrimination ratio is at least a few thousand times in favor of the monovalent cation. A major part of this discrimination is caused by the very low mobility of the divalent cation within the membrane compared with that of the monovalent cation. The remainder of the discrimination is caused by the selectivity of the membranes which prefer monovalent to divalent cations. There is a clear discrepancy between Ba⁺⁺ diffusibility and mobility within, the membrane. This implies that Ba⁺⁺ may move within the hydrophobic membrane as a neutral complex. Some similarity with natural biological membranes is indicated.     

Effects of Cations on Antimicrobial Activity of Ostricacins-1 and 2 on <Emphasis Type="Italic">E. coli</Emphasis> O157:H7 and <Emphasis Type="Italic">S. aureus</Emphasis> 1056MRSA

Ostricacin-1 and ostricacin-2 (Osp-1 and Osp-2) were β-defensins antimicrobial peptides that were purified from ostrich leukocytes using a cation-exchange column and a semi-prep RP-HPLC column. Both ostricacins were subjected to increased concentrations of monovalent cations (K⁺ and Na⁺) and divalent cations (Ca²⁺ and Mg²⁺) in order to investigate the effect of cations on the activity of these ostricacins on Gram-negative bacteria and Gram-positive bacteria. The radial diffusion assay method showed that both ostricacins were sensitive to the presence of cations. The divalent cations showed more antagonized effect on the activity against Gram-negative bacteria than the monovalent cations, as the ostricacins lost ability to inhibit bacterial growth at very low concentration (5 mM). When viewed in the context of other defensins activity, our data support a hypothesis that defensins’ overall net positive charge determine the sensitivity to cations.     

Mono- and divalent competitive adsorption to a charged membrane in a closed system: A comparative study

Competitive adsorption to a negative surface between monovalent and divalent cations is studied in a closed system. A self-consistent theory is presented for the cases when the divalent cation binds to two negative sites (1:2 binding) and to one negative site (1:1 binding). It is demonstrated that these two cases are distinguishable when the relative difference in total divalent concentrations determined at fixed bulk divalent concentrations is plotted as a function of the bulk monovalent concentration. The 1:2 binding case yields a sigmoidal curve while the 1:1 binding curve is hyperbolic. The comparison between the 1:2 and 1:1 binding cases of the divalent cation is extended to include; (1) the existence of a surface charge to which no binding occurs, and (2) the case when an additional non-binding monovalent cation is present.     

Uridine uptake inhibition in Novikoff hepatoma cells by perturbants of intracellular Ca2+ and K+ levels

The rate of uptake of uridine into the acid-soluble fraction of Novikoff hepatoma cells is inhibited by low concentrations of the ionophores A23187 and gramicidin and other perturbants of intracellular cation levels. Inhibition of uridine uptake by A23187 is dependent on Ca2+ and is reduced by serum and high levels of Mg2+. The effectiveness of A23187 is dependent on the Ca2+/Mg2+ ratio rather than the absolute concentration of either ion. Inhibition of uridine uptake by gramicidin is not significantly affected by serum or divalent cations. Other effectors of monovalent cation flux such as ouabain and valinomycin also inhibit uridine uptake. These results indicate that net uptake of uridine may be influenced by intracellular levels of certain monovalent and divalent inorganic cations.     

Effect of membrane surface charge on nickel uptake by purified mung bean root protoplasts

The influence of membrane surface charge on cation uptake was investigated in protoplasts prepared from roots of mung bean (Vigna radiata L.). Confocal laser scanning microscopy showed that a fluorescent trivalent cation accumulated to very high concentrations at the surface of the protoplasts when they were incubated in medium containing low concentrations of Ca or other cations, but that this accumulation could be completely reversed by suppression of membrane surface negativity by high cation concentrations. Influx of 63Ni was strongly reduced by a range of divalent cations. Increasing the Ca concentration in the medium from 25 microM to 10 mM inhibited 63Ni influx by more than 85%. 63Ni influx was also inhibited by 85% by reducing the pH from 7 to 4. Computation of the activity of Ni at the membrane surface under the various treatment conditions showed that Ni uptake was closely correlated with its activity at the membrane surface but not with its concentration in the bulk medium. It was concluded that the effects on Ni uptake of addition of monovalent, divalent and trivalent cations, and of variations in pH are all consistent with the proposition that the activity of Ni at the membrane surface is the major determinant of the rate of Ni influx into mung bean protoplasts. It is proposed that the surface charge on the plasma membrane will influence the membrane transport of most charged molecules into cells.     

Conductometric analysis of the competition between monovalent and divalent counterions in their interaction with polyelectrolytes

A procedure is described for the analysis of the conductivity of solutions of anionic polyelectrolytes in which both mono- and divalent counterions are present. The method is based on analysis of the relation between the overall conductivity of the system and the conductivity of the individual monovalent cations which are only electrostatically (non-specifically) bound. The system is described in terms of the two-state approach, implying that the counterions are considered to be either fully bound to the polyion or completely free. The potentialities of the proposed method are explored by studying solutions of alkali polyacrylates with and without added zinc nitrate at several alkali nitrate concentrations. The results give a picture of the composition of the counterionic atmosphere around the polyion in systems with both mono- and divalent counterions present. To a certain degree, the divalent ion Zn(II) was found to be bound quantitatively by the polyion. The composition of the counterionic atmosphere around the polyion was largely independent of alkali nitrate concentration when the latter was present in not too large an excess with respect to both Zn(II) and the charged monomers.     

A deoxyribonucleic acid ligase from nuclei of rat liver. Purification and properties.

A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.