The interaction of sodium alginate with univalent cations

The M/G ratio, dyad and triad frequencies in the sodium alginate chain, were determined from ¹³C-nmr spectra. The interactions of sodium alginate in solution with the univalent cations K⁺ ion and Na⁺ ion have been investigated by viscometry and membrane osmometry. The dependencies of intrinsic viscosity, Huggins constant, and second virial coefficient on ionic strength were observed, and the maximums in reduced viscosity were obtained in low KCl and NaCl concentrations, respectively. These show that the electroviscous effects play an important role in polyelectrolyte solution, and the effect of the Na⁺ ion on aqueous solution of sodium alginate is greater than the K⁺ ion. The experimental observations are interpreted in terms of ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues and cooperation “egg-box” binding between polyguluronate chain sequence. The difference of interaction between univalent cations and alginate chains in solution is attributed to the ability of their binding with the polyion, which depends on the properties of ions itself. © 1998 John Wiley & Sons, Inc. Biopoly 46: 395–402, 1998     

Influence of Certain Cations on Activity of Succinyl CoA Synthetase From Tobacco

Succinyl CoA synthetase from Nicotiana tabacum exhibited a requirement for univalent and divalent cations. Mn²⁺ replaced Mg²⁺ in the assay medium and Co²⁺ and Ca²⁺ partially replaced Mg²⁺. Addition of Zn²⁺ resulted in no enzyme activity. The enzyme was activated by univalent cations K⁺, Rb⁺, NH₄⁺, and Na⁺; Li⁺ showed little or no activation. Maximum enzyme activity varied significantly with potassium salts of different anions. Greatest activation was obtained with K₃PO₄ and, respectively, KCl, KNO₃, K₂SO₄ and KF exhibited steadily decreasing enzyme activation.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

Alkali cation selectivity of the wheat root high-affinity potassium transporter HKT1

The wheat root high-affinity K⁺ transporter HKT1 functions as a sodium-coupled potassium co-uptake transporter. At toxic millimolar levels of sodium (Na⁺), HKT1 mediates low-affinity Na⁺ uptake while potassium (K⁺) uptake is blocked. In roots, low-affinity Na⁺ uptake and inhibition of K⁺ uptake contribute to Na⁺ toxicity. In the present study, the selectivity among alkali cations of HKT1 expressed in Xenopus oocytes and yeast was investigated under various ionic conditions at steady state. The data show that HKT1 is highly selective for uptake of the two physiologically significant alkali cations, K⁺ and Na⁺ over Rb⁺, Cs⁺ and Li⁺. In addition, Rb⁺ and Cs⁺, and an excess of extracellular K⁺ over Na⁺, are shown to partially reduce or block HKT1-mediated K⁺-Na⁺ uptake. Furthermore, K⁺, Rb⁺ and Cs⁺ also effectively reduce outward currents mediated by HKT1, thereby causing depolarizations. In yeast, HKT1 can produce high-affinity Rb⁺ uptake at approximately 15-fold lower rates than for K⁺. Rb⁺ influx in yeast can be mediated by the ability of the yeast plasma membrane proton pump to balance the 35-fold lower HKT1 conductance for Rb⁺. A model for HKT1 activity is presented involving a high-affinity K⁺ binding site and a high-affinity Na⁺ binding site, and competitive interactions of K⁺, Na⁺ and other alkali cations for binding to these two sites. Possible implications of the presented results for physiological K⁺ and Na⁺ uptake in plants are discussed.     

Induction of Myxospore Formation in Stigmatella aurantiaca (Myxobacterales) by Monovalent Cations

Suspension cultures of Stigmatella aurantiaca can be induced to form myxospores by addition of the monovalent cations Li⁺, Na⁺, NH₄⁺, K⁺, or Rb⁺.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Molecular analysis of the mechanism of potassium uptake through the TRK1 transporter of Saccharomyces cerevisiae

The TRK-HKT family of K⁺ transporters mediates K⁺ and Na⁺ uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K⁺ or Rb⁺, both of which bind the two binding sites of ScTRK1 with very high affinities in K⁺-starved cells. Na⁺ can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K⁺ and Na⁺, or Rb⁺ and Na⁺, the transporter takes up Na⁺ together with K⁺ or Rb⁺. Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln⁹⁰ is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu⁹⁴⁹ is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K⁺ uptake. This is not Na⁺-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K⁺-H⁺ cotransport.     

Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes

A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb⁺ > Cs⁺ > Na⁺ > K⁺ > Li⁺. The conductance of the membrane was increased up to a value of about 10⁻² ohm⁻¹/cm² with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn²⁺ > Cd²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺.     

Activation of Rb+ and Na+ uptake into yeast by monovalent cations

⁸⁶Rb⁺ uptake by yeast was not only stimulated by Rb⁺ or K⁺ but also by Na⁺. The uptake of ²²Na⁺ was enhanced by both Rb⁺ and K⁺, but not by Na⁺, which was inhibitory at all concentrations applied. Inhibition of ²²Na⁺ uptake by inactive Na⁺ occurred in two phases: one phase refers to inhibition at low Na⁺ concentrations and the other to inhibition at high Na⁺ concentrations. Our results can be qualitatively described by a two-site transport mechanism, having two cation binding sites, which must be occupied with monovalent cations before transport can occur.     

BIOELECTRIC POTENTIALS IN VALONIA : II. EFFECTS OF ARTIFICIAL SEA WATERS CONTAINING LiCl,CsCl, RbCl,OR NH4Cl

In their influence on the P.D. across the protoplasm of Valonia macrophysa, Kütz., Li⁺ and Cs⁺ resemble Na⁺, while Rb⁺ and NH₄ ⁺ resemble K⁺. The apparent mobilities of the ions in the external surface layer of Valonia protoplasm increase in the order: Cs⁺, Na⁺, Li⁺ < Cl^- < Rb⁺ < K⁺ < NH₄ ⁺.     

Characterisation of cation binding and gelation of polyuronates by circular dichroism

The cation-induced gelation of alginates and pectins with various metal ions has been monitored by circular dichroism (c.d.), using a controlled diffusion technique to prepare homogeneous gels in situ. Spectral changes observed with Ca²⁺ are closely similar to those previously reported for Ca²⁺-induced dimerisation of alginate poly-l-guluronate and pectin poly-d-galacturonate chain-sequences in solution, and the magnitude of the c.d. change on gel formation is directly related to the proportion of these structural types present. It therefore appears that gel formation does not introduce optical artefacts such as have been reported for particulate systems or biological membranes. Similar spectral changes are observed on gelation of pectin with Sr²⁺, Ba²⁺, Cd²⁺, Ni²⁺, or Pb²⁺, but with minor alterations in the wavelength of maximum c.d. change. These subtle differences are interpreted as reflecting variation in binding-site geometry to accommodate ions of different size. Differences in c.d. behaviour with Mg²⁺, Ca²⁺, and Sr²⁺ are far greater for alginate than for pectin, consistent with the greater selectivity of ion-binding. Gelation of both alginate and pectin with Cu²⁺ is accompanied by spectral changes that are opposite in sign to those observed with other divalent cations, and span a much wider range of wavelengths. This suggests a different and less-specific binding mechanism, consistent with the known lack of selectivity of Cu²⁺ for different polyuronates. However, for alginate, there is also evidence of some specific interchain chelation. A minor enhancement of alginate c.d. in the presence of K⁺ ions is attributed to a decrease in charge density of the polymer chain by bound cations, with consequent increase in segment-segment association in solution. The sign and magnitude of this effect confirm the selectivity of polyuronates for divalent cation.     

E2→E1 transition and Rb+ release induced by Na+ in the Na+/K+-ATPase. Vanadate as a tool to investigate the interaction between Rb+ and E2

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb⁺ deocclusion stimulated by Na⁺ in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb⁺ and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb⁺ deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb⁺]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb⁺-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na⁺-stimulated Rb⁺ deocclusion would require the release of at least one Rb⁺ ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb⁺ in equilibrium conditions. Results show that, while Mg^2 + decreases the affinity for Rb⁺, addition of vanadate offsets this effect, increasing the affinity for Rb⁺. In transient experiments, we investigated the exchange of Rb⁺ between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na⁺ without significantly affecting the rate of Rb⁺ deocclusion. On the other hand, we found the first evidence of a very low rate of Rb⁺ occlusion in the enzyme–vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb⁺.     

Direct NMR detection of the "invisible" alkali metal cations tightly bound to G-quadruplex structures

We report the first direct solution NMR detection of the alkali metal cations (²³Na⁺, ³⁹K⁺, and ⁸⁷Rb⁺) residing inside G-quadruplex channel structures formed by guanosine 5′-monophosphate and a DNA oligomer, d(TG₄T). In solution, these channel alkali metal cations are tightly bound to the G-quadruplex structure and have been considered to be “invisible” to NMR spectroscopy for many years. Our finding that it is possible to directly observe these alkali metal cations by NMR spectroscopy provides a new tool for studying cation binding affinity and dynamics in G-quadruplex DNA.     

Biochimica et biophysica acta,vol. 644 (1981)

In the presence of an iso-osmotic concentration (0.4 M) of LiCl, the exit of cellular K⁺ and concomitant entry of Li⁺ in the marine bacterium, Vibrio alginolyticus, were enhanced by an increase in the medium pH, with an optimum at about pH 9.6. In addition to alkaline pH, the K⁺ exit in the NaCl medium required the presence of a weak base such as diethanolamine, ethanolamine or methylamine, which is permeable to the membrane in its unprotonated form. No net entry of Na⁺ was detected in this case and the amine accumulated in exchange for K⁺. The K⁺ exit observed at alkaline pH could be explained by the function of a K⁺/H⁺ antiporter. Once the cells were loaded with the amine, their exposure to the NaCl medium in the absence of loaded amine induced the entry of Na⁺. In RbCl or CsCl medium, fast entry of Rb⁺ or Cs⁺ and exit of K⁺ were observed at neutral pH (7.5), and the rate of K⁺ exit increased with the medium pH. From these results, we established a simple method for the replcement of cellular cations with a desired cation (Li⁺, Na⁺, K⁺, Rb⁺ or Cs⁺). The present method was found to be applicable also to Escherichia coli.     

Effects of monovalent cations on the catalytic activity of pig liver phosphofructokinase.

The monovalent cations NH₄⁺, K⁺, and Rb⁺ activate pig liver phosphofructokinase by increasing the maximal velocity. In the presence of these cations the enzyme retains sigmoid kinetics with respect to fructose-6-phosphate. However, these cations bring about a decrease in the [S]_0.5 for fructose-6-phosphate to an extent directly proportional to their ionic volumes. The apparent dissociation constants of NH₄⁺, K⁺, and Rb⁺ for the enzyme at 0.5 mM ATP and 4 mM Fru6P are 0.2 mM, 8 mM, and 15 mM, respectively. The maximal velocity of the enzyme in the presence of saturating concentrations of Rb⁺ is about 70% of that seen with NH₄⁺ or K⁺. The monovalent cations Li⁺, Na⁺, and Cs⁺ inhibit the enzyme at high concentrations (> 50 mM) by decreasing the maximal velocity. Although the efficiency of inhibition by these cations qualitatively increases with decreasing size, there is no obvious quantitative relationship between efficiency of inhibition and any parameter of ionic size.     

Chiroptical characterisation of polysaccharide secondary structures in the presence of interfering chromophores: Chain conformation of inter-junction sequences in calcium alginate gels

The changes in chain conformation which accompany Ca²⁺-induced gelation of alginate have been investigated by a combined circular dichroism (c.d.) and optical rotatory dispersion (o.r.d.) approach. C.d. changes in the carboxyl n→π^* spectral region, arising predominantly from formation of calcium poly-l-guluronate junctions, were monitored for three alginates of widely differing block composition. The corresponding o.r.d. changes, calculated by Kronig-Kramers trnasform, were subtracted from the observed changes in o.r.d. on gelation, to “unmask” the changes in optical activity of the conformation-sensitive electronic transitions of the polysaccharide backbone. Contributions to the “residual” o.r.d. difference spectra from poly-l-guluronate, poly-d-mannuronate, and heteropolymeric chain-sequences were calculated by solution of simultaneous equations at each wavelength. Results for poly-guluronate sequences are in agreement with previous studies of alginate films by vacuum ultraviolet c.d., and with observed c.d. and o.r.d. changes on addition of calcium ions to homopolyguluronate segments in solution. The much greater changes in backbone optical activity calculated for polymannuronate and heteropolymeric chain-sequences, however, have no counterpart in the behaviour of these sequences in isolation. An explanation is proposed in terms of stretching of interconnecting sequences between calcium polyguluronate junctions in alginate gels, to give a more-extended chain conformation than in free solution.     

The penetration of some cations into muscle

The influx of Na⁺, K⁺, Rb⁺, and Cs⁺ into frog sartorius muscle has been followed. The results show that a maximum rate is found for K⁺, while Na⁺ and Cs⁺ penetrate much more slowly. Similar measurements with Ca⁺⁺, Ba⁺⁺, and Ra⁺⁺ show that Ba⁺⁺ penetrates at a rate somewhat greater than that of either Ca⁺⁺ or Ra⁺⁺. All these divalent cations, however, penetrate at rates much slower than do the alkali cations. The results obtained are discussed with reference to a model that has been developed to explain the different penetration rates for the alkali cations.     

Ion Specificity of Na+-Transporting Systems in the Plasma Membrane of the Halotolerant Alga Tetraselmis (Platymonas) viridis

Vesicular preparations of plasma membranes (PM) from the microalga Tetraselmis (Platymonas) viridisRouch were used to investigate the ion specificity of the Na⁺/H⁺antiporter and Na⁺-translocating ATPase, two Na⁺-transporting systems previously identified functionally by our studies of T. viridisPM. The Na⁺/H⁺antiporter and Na⁺-ATPase were shown to translocate, with similar efficiencies, Na⁺and Li⁺across the membrane, whereas other cations, such as K⁺, Rb⁺, and Cs⁺, were not transported by these systems. Transport of the latter cations across PM of T. viridisoccurred through the ion channels of PM, which were apparently selective for K⁺.     

Physical and kinetic properties of sheep liver pyridoxine kinase

Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn²⁺ > Mn²⁺ > Mg²⁺. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn²⁺ > Zn²⁺ > Mg²⁺. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K⁺, then Rb⁺ and NH₄⁺, whereas the enzyme had very little activity with Na⁺, Li⁺, or Cs⁺. Na⁺ did not interfere with the activation by K⁺. The activation of the kinase by K⁺, NH₄⁺, and Rb⁺ followed Michaelis-Menten kinetics, and the apparent K_m values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent K_m value of the enzyme for pyridoxine and had little or no effect on the K_m for ZnATP^2? or the V of the kinase-catalyzed reaction.