Binding of cations by microsomes from rabbit skeletal muscle

Fragmented sarcoplasmic reticulum and transverse tubular system, as isolated in the microsomal fraction from rabbit skeletal muscle, bind H⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and Zn⁺⁺. The binding depends on a cation exchange type of interaction between these cations and the chemical components of the membranous systems of the muscle cell. The monovalent and divalent cations exchange quantitatively for each other at the binding sites on an equivalent basis. Scatchard plots of the H⁺ binding data indicate that the binding groups can be resolved into two major components in terms of their pK values. Component 1 has a pK value of 6.6 and a capacity for H⁺ binding of 2.2 meq/g N . The second component has a much higher H⁺ binding capacity (7–8 meq/g N ), but its pK value, 3.4, is non-physiological. The binding of cations other than H⁺ at a neutral pH occurs at the binding sites making up component 1. The order of affinity of the cations for the microsome binding sites is H⁺ » Zn⁺⁺ > Ca⁺⁺ > Mg⁺⁺ » Na⁺ = K⁺ as reflected by the apparent respective pK_M values: 6.6, 5.2, 4.7, 4.2, 1.3, 1.3. Caffeine, which causes contracture and potentiates the twitch of skeletal muscle, does not interfere with the binding of Ca⁺⁺ by the microsomes at neutral pH.     

Selectivity of interaction of univalent cations with mammalian ribosomes studied by equilibrium dialysis in the presence of the K+ analogue, 204Tl+

The interaction of K⁺ with mammalian ribosomes was studied by equilibrium dialysis and compared with that of other univalent cations. The heavy K⁺ analogue, Tl⁺, binds more firmly than K⁺ to ribosomes and, unlike K⁺, has a practically useful isotope. With ²⁰⁴Tl⁺ as a marker of K⁺-selective binding the ribosome-cation interaction could be followed down to levels below 0.1 average Tl⁺-occupied site per ribosome. The Tl⁺/ribosome ratio varied with the free Tl⁺ concentration in a multiple way. At high Tl⁺ saturation Tl⁺ was easily displaced by Mg²⁺. With decreasing Tl⁺ saturation the competitive activity of Mg⁺⁺ was strikingly reduced, indicating that Tl⁺ and Mg⁺⁺ compete with different efficiency for different classes of sites.The experiments on univalent cations were performed at 1.5 mM Mg²⁺ under two complementary conditions: (1) Ribosomes were pretreated with 5 × 10^?2, 5 × 10^?3, and 5 × 10^?4 M LiNO₃, NaNO₃, KNO₃, and CsNO₃, and then equilibrated with different concentrations of ²⁰⁴TlNO₃ in the same buffers. (2) Ribosomes were pretreated with 10^?2, 10^?4, and 10^?6 M ²⁰⁴TlNO₃, and then equilibrated with different concentrations of LiNO₃, NaNO₃, KNO₃, and CsNO₃ (displacement experiments). At high Tl⁺ saturation Na⁺ and Li⁺ were about as active as K⁺ and Cs⁺ in competing with ²⁰⁴Tl⁺. With decreasing Tl⁺ saturation a differentiation occurred in favor of K⁺ and Cs⁺, with some preference for K⁺. It is concluded that ribosomes contain a limited number of sites with pronounced ion specificity. Of physiological cations K⁺ is most firmly bound to these sites.     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

In the E₁ state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di- and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br₂-Titu³⁺ is bound with the highest affinity (<μm), among the tested divalent cations Ca²⁺ binds the strongest, and Na⁺ binds with about the same equilibrium dissociation constant as Mg²⁺ (∼0.8 mm). For alkali ions it exhibits binding affinities following the order of Rb⁺≃ K⁺ > Na⁺ > Cs⁺ > Li⁺. The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na⁺ ions the equilibrium dissociation constant is < 0.01 mm. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na⁺, no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mm. Received: 7 August 2000/Revised: 14 November 2000     

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.     

Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

Lanthanide/proline cotransport across rabbit renal brush borders

Summary It has been suggested previously that La³⁺ can replace Na⁺ on various cotransport systems in renal brush border membranes. In the present study, we used rabbit renal brush border membrane vesicles to examine the specificity and kinetics of Ln³⁺/proline cotransport. Experiments were carried out under zero-trans, voltage clamped conditions using a rapid-mix/filtration technique. Initial experiments confirmed that La³⁺ produced the classical overshoot phenomenon. The initial rates of proline uptake relative to Na⁺ were Eu³⁺, Tb³⁺, Nd³⁺, Pr³⁺, Ho³⁺ (3.3)>Na⁺ (1.0)>La³⁺ (0.86) > choline⁺ (0.1). At a saturating salt concentration, uptake saturated with increasing proline concentration: theK _t andJ _max were 0.05mm and 17 pmol mg^–1 sec^–1 in Na⁺; and 0.28mm and 73 pmol mg^–1 sec^–1 in Tb³⁺. The higherJ _max in Tb³⁺ indicates that the Tb³⁺-proline loaded carrier is more effective than the Na⁺-proline loaded carrier in overcoming some rate-limiting barriers in the transport process. Na⁺ activated proline uptake with a Hill coefficient of 1.6 and aK _0.5 of 21mm, while Tb³⁺ activated with a Hill coefficient of 0.88 and aK _0.5 of 28mm. The Hill coefficient for Na⁺ suggests two binding sites, whereas the Hill coefficient for Tb³⁺ may indicate negative cooperativity between the trivalent ligands at the binding sites. We conclude that lanthanides are able to substitute for Na⁺ on the brush border proline carrier and that the lanthanides may serve as useful probes for the ligand binding sites.     

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.     

Interaction of alginate single-chain polyguluronate segments with mono- and divalent metal cations: a comparative molecular dynamics study

The interaction of a set of monovalent (Na⁺, K⁺) and divalent (Mg²⁺, Ca²⁺) metal cations with single-chain polyguluronate (periodic chain based on a dodecameric repeat unit, 2₁-helical conformation) is investigated using explicit-solvent molecular dynamics simulations (at 300 K and 1 bar). A total of 14 (neutralising) combinations of the different ions are considered (single type of cation or simultaneous presence of two types of cation, either in the presence or absence of chloride anions). The main observations are: (1) the chain conformation and intramolecular hydrogen bonding is insensitive to the counter-ion environment; (2) the binding of the cations is essentially non-specific for all ions considered (counter-ion atmosphere confined within a cylinder of high ionic density, but no well-defined binding sites); (3) the density and tightness of the distributions of the different cations within the counter-ion atmosphere follow the approximate sequence Ca²⁺>Mg²⁺>K⁺～Na⁺; (4) the solvent-separated binding of the cations to the carboxylate groups of the chain is frequent, and its occurrence follows the approximate sequence K⁺>Na⁺>Ca²⁺>Mg²⁺ (contact-binding events as well as the binding of a cation to multiple carboxylate groups are very infrequent); and (5) the counter-ion atmosphere typically leads to a complete screening of the chain charge within 1.0–1.2 nm of the chain axis and, for most systems, to a charge reversal at about 1.5 nm (i.e. the effective chain charge becomes positive at this distance and as high in magnitude as one-quarter of the bare chain charge, before slowly decreasing to zero). These findings agree well (in a qualitative sense) with available experimental data and predictions from simple analytical models, and provide further insight concerning the nature of alginate–cation interactions in aqueous solution.     

Specific ion effects on the solution conformation of poly-L-proline

The effects of various electrolytes on the conformation of poly-L -proline II in aqueous and nonaqueous solution have been investigated by optical rotatory techniques. It is shown that these agents induce a linear decrease in the corrected specific levorotaton of poly-L -proline with increasing salt concentration, with a molar effectiveness which varies from one salt to another. The salt-induced rotatory changes may be divided into anion and cation components, and it is shown that the major specific affectors are the anions, which increase in effectiveness in reducing the corrected specific levorotation in the following sequence: Cl^? < NO₃^? < Br^? < I^? < ClO₄^? < SCN^?. The inorganic monovalent cations tested (Li⁺, Na⁺, K⁺) are all equally effective in decreasing the specific levorotation. Ca⁺⁺ has a marginally greater effect per mole than the inorganic monovalent cations, while the effectiveness of the ganidinium cation is appreciably less. The tetraalkylammonium cations decrease the specific levorotation more effectively than the inorganic monovalent cations, with the molar effectiveness increasing linearly with total content of methyl plus methylene groups. A similar linear increase with increasing methyl plus methylene content is shown by the aliphatic alcohols, though the effect per mole of CH₂ or CH₃ group is appreciably smaller than that shown by the tetraaklylammonium cations. Salts dissolved in essentially anhydron for mamide are also appreciably effective. Selected viscosity experiments have also been carried out to show that the observed effects on specific levorotation have a structural as well as an optical basis. These results are interpreted in terms of a model which involves binding of anions at the imide nitrogen, and cations at the carbonyl oxygen. It is proposed that this binding induces an increase in the double-bond character; of the peptide bond (and thus a shortening of the bond) which is roughly proportional in the polarizability of the bound anion and that this increase is potentiated by cations which decrease the total dielectric constant (e.g., the tetraalkylammonium series), and reduced by cations presenting competitive local anion binding sites (e.g., the guanidinium ion). We propose further that this shortening of the peptide bond is accompanied by a lengthening of the adjacent bond, thus reducing the steric restraints to rotation about this bond (increasing the accessible range of the angle ψ) sufficiently to induce a progressive non-cooperative collapse of the poly-L -proline II structure. Several lines of evidence are presented to support this interpretation. The various neutral salts are also shown to induce a time-dependent precipitation or “salting-out” of poly-L -proline from solution. In order of decreasing molar effectiveness as salting-out agents in this system, the various ions may be ranked: SO₄^? > Ac^? > Cl^? > Br^? > SCN^? > I^? > ClO₄^?; and K⁺ ? Na⁺ > Li⁺ > Ca⁺⁺. These rankings follow the usual Hofmeister or lyotropic series, and are quite different from hose which apply to the effects on solution conformation of poly-L -proline.     

Kinetic studies of a (Na++ K++ Mg2+)ATPase in sugar beet roots. III. A proposed model for the (Na++ K+) activation and its significance for field properties

A microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase preparation from sugar beet roots was used. The activation by simultaneous addition of Na⁺ and K⁺ at different levels was examined in terms of steady state kinetics. The observed data can be summarized in the following way: 1. The apparent affinity between the enzyme and the substrate MgATP depends on the ratio between Na⁺ and K⁺. At low Na⁺ concentration (below 5 mM), the apparent K_m decreases with increasing concentrations of K⁺ (1–20 mM). At 5 mM Na⁺, the K⁺ level does not change the apparent K_m, while at Na⁺ levels above 10 mM, the apparent K_m between enzyme and substrate increases with increasing concentration of K⁺. 2. When the MgATP concentration is kept constant, homotropic cooperativity (concerning one type of ligand) and heterotropic cooperativity (concerning different types of ligands) exist in the activation by Na⁺ and K⁺. The Na⁺ binding is cooperative with different K_m values and Hill coefficients (n) in the presence of low and high concentration of K⁺. At low Na⁺ level (< 5 mM). a negative cooperativity exists for Na⁺ (n_Na < 1) which is more pronounced in the presence of high [K⁺]. When the concentration of Na⁺ is raised the negative cooperativity disappears and turns into a positive one (n_Na > 1). Only K⁺ binding in the presence of low [Na⁺] shows cooperativity with a Hill coefficient that reflects changes from negative to positive homotropic cooperativity with increasing concentrations of K⁺ (n_K < 1 → n_K > 1). In the presence of [Na⁺] > 10 mM, the changes in n_k are insignificant. 3. A model is proposed in which one or two different K sites and one or two Na sites control the catalytic activity, with multiple interactions between Na⁺, K⁺ and MgATP. 4. In the presence of Na⁺ (< 10 mM), K⁺ is probably bound to two K sites, one of which translocates K⁺ through the membrane by an antiport Na⁺/K⁺ mechanism. This could be connected with an elevated K⁺ uptake in the presence of Na⁺ and could therefore explain some field properties of sugar beets.     

Modeling of the Interaction of Na+ and K+ with the Binding of the Cocaine Analogue 3β-(4-[125I]Iodophenyl)tropane-2β-Carboxylic Acid Isopropyl Ester to the Dopamine Transporter

Abstract: The present study examines the interaction of Na⁺ and K⁺ with the binding of the cocaine analogue 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylic acid isopropyl ester to dopamine transporters (DATs) in rat striatal synaptosomal membranes at 37°C. The binding increases with [Na⁺] from 10 to 100 mM and decreases with higher [Na⁺]. The presence of K⁺ reduces the maximal stimulatory effect of Na⁺ and causes a nonlinear EC₅₀ shift for Na⁺. K⁺ strongly inhibits the binding at low [Na⁺]. Increasing [Na⁺] produces a linear IC₅₀ shift for K⁺. Saturation analysis indicates a single binding site changing its affinity for the radioligand depending on [K⁺]/[Na⁺] ratio in the assay buffer. A reduced B_max was observed in the presence of 10 mM Na⁺ and 30 mM K⁺. Both high [Na⁺] and high [K⁺] accelerate the dissociation of the binding, and K⁺-induced acceleration was abolished by increasing [Na⁺]. Least squares model fitting of equilibrium data and kinetic analysis of dissociation rates reveal competitive interactions between Na⁺ and K⁺ at two sites allosterically linked on the DAT: One site mediates the stimulatory effect of Na⁺, and the other site involves the radioligand binding and the inhibitory effect of cations on the binding. Various uptake blockers and substrates, dopamine in particular, display reduced potency in inhibiting the binding at a higher [K⁺]/[Na⁺] ratio.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

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.     

Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes

The additional activation by monovalent cations of the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-dependent}$ ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca²⁺-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca²⁺ activation is characterized by low apparent Ca²⁺ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca²⁺ affinity and high maximum activity.At optimum concentrations of Ca²⁺ the additional activation of the B-state by K⁺, NH₄⁺, Na⁺ and Rb⁺ exceeded the corresponding activations of the A-state, and half-maximum activations by K⁺, NH₄⁺, and Na⁺ were achieved at lower concentrations in the B-state than in the A-state. Li⁺ and Cs⁺ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order $\text{K}{}^{\mathrm{+}}\text{> NH}{}_4{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{= Rb}{}^{\mathrm{+}}\text{> Li}{}^{\mathrm{+}}\text{= Cs}{}^{\mathrm{+}}$. The A-state was activated almost equally by K⁺, Na⁺, NH₄⁺, and Rb⁺ and to a smaller extent by Li⁺ and Cs⁺.At sub-optimum concentrations of Ca²⁺ high concentrations of monovalent cations (100 mM) activated the Ca²⁺-ATPase equally in the A-state and the B-state. In the absence of Ca²⁺ the monovalent cations inhibited the Mg²⁺-dependent ATPase in both types of membranes. This dependence on Ca²⁺ indicates that the monovalent cations interact with the Ca²⁺ sites in the B-state.The results suggest that K⁺ or Na⁺, or both, contribute to the regulation of the Ca²⁺ pump in erythrocytes.     

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.     

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase

(1) The fluorescence of eosin Y in the presence of (Na⁺ + K⁺)-ATPase is enhanced by Mg²⁺. The enhancement by Mg²⁺ is larger than that obtained with Na⁺ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg²⁺ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na⁺. (2) The Mg²⁺-dependent enhancement of fluorescence can be reversed by K⁺ as well as by ATP. In the presence of Mg²⁺ + P_i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg²⁺ and a low concentation of K⁺ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg²⁺ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K⁺-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per ³²P-labelling site in the presence of 5 mM Mg²⁺ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg²⁺ + 0.25 P_i, the K_d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg²⁺, 150 mM Na⁺, the K_d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg²⁺, the addition of K⁺ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per ³²P-labelling site). With 5 mM Mg²⁺ + 150 mM K⁺, the affinities of the two binding sites become identical, at a K_d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg²⁺-form to the K⁺-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na⁺ in the presence of Mg²⁺ does not appreciably change the rate of conversion to the K⁺-form, giving a rate constant of about 110 s^?. However, the addition of oligomycin in the presence of Mg²⁺ + Na⁺ had a profound effect: the rate of conversion to the K⁺-form was decreased by a factor of 2000 to about 0.063 s^?1. This suggests that the conformation with Mg²⁺ alone is different from the conformation with Na⁺ alone. (6) The effects of K⁺, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per ³²P-labelling site are bound to ATP sites.     

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.     

Cation requirement for the reconstitution of oligomycin-sensitive ATPase by means of soluble F1-ATPase and F1-depleted submitochondrial particles

Bovine heart submitochondrial particles depleted of F₁ by treatment with urea (‘F₁-depleted particles’) were incubated with soluble F₁-ATPase. The binding of F₁ to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH⁺₄, K⁺, Rb⁺, Cs⁺, Na⁺ and Li⁺ promoted reconstitution maximally at 40–74 mM, guanidinium⁺ and Tris⁺ at 20–30 mM, and Ca²⁺ and Mg²⁺ at 3–5 mM. The particles exhibited a negative ζ-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F₁ binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F₁-depleted thylakoid membranes and with submitochondrial particles depleted of both F₁ and the coupling proteins F₆ and oligomycin sensitivity-conferring protein.     

Response of sugarcane to different types of salt stress

Summary Due to climatic conditions and prevailing water regime the yield and sucrose recovery in sugarcane are high in South Western India. However, excessive irrigation, poor drainage and luxuriant use of fertilizers have resulted in conversion of large fertile areas into saline lands. The salinity is due to the excess of Na⁺, Ca⁺⁺, Mg⁺⁺, SO₄ ^– and Cl^– ions. Individual salts of NaCl, Na₂SO₄, MgCl₂ and MgSO₄ were employed in culture experiments to study salt stress effect on sugarcane variety Co 740. It was observed that sulphate salinity was more toxic to sugarcane than the chloride one. Sulphate salts caused more inhibition of growth, chlorophyll synthesis, PEPCase activity, decreased the uptake of K⁺ and Ca⁺⁺ ions but stimulated nitrate reductase. The stress did not result in proline accumulation in the sugarcane cultivar Co 740. The degree of toxicity of different ions in decreasing order in sugarcane cultivar Co 740 is SO₄ ^–>Na⁺>Cl^–>Mg⁺⁺.