Cation Penetration through Isolated Leaf Cuticles

The rates of penetration of various cations through isolated apricot Prunus armeniaca L. leaf cuticles were determined. Steady state rates were measured by using a specially constructed flow-through diffusion cell. The penetration rates of the monovalent cations in group IA followed a normal lyotropic series, i.e., CS⁺ ≥ Rb⁺ > K⁺ > Na⁺ > Li⁺. The divalent cations all penetrated through the cuticle more slowly than the monovalent cations. Comparison of the relative values of k (permeability coefficient) and D (diffusion coefficient) indicates that the penetration of ions through isolated cuticles took place by diffusion and was impeded by charge interactions between the solute and charge sites in the penetration pathway. Cuticular penetration rates of K⁺ and H₂O at pH above 9 were of similar magnitude. At pH 5.5 H₂O penetration was not affected but that of K⁺ was greatly reduced. From this observation and from data on cuticle titration and ion adsorption studies, we hypothesize that cuticular pores are lined with a substance (perhaps a protein) which has exposed positively charged sites.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Activation of diol dehydrase by formamidinium or guanidinium ion, polyatomic monovalent cations having sp2 nitrogen

Coenzyme B₁₂-dependent diol dehydrase was activated by formamidinium or guanidinium ion. These polyatomic monovalent cations having sp² hybrid atomic orbitals and trigonal orientation were much more effective in activating the enzyme than methylammonium ion, but less active than NH₄+ or K⁺. Formamidinium and guanidinium ions were also effective both in forming and maintaining the binding of coenzyme B₁₂ to the apoenzyme. There is a close relationship between the effectiveness in activating the enzyme and those in forming and maintaining the holoenzyme, suggesting that these polyatomic monovalent cations play the same role in the diol dehydrase system as alkali metal monovalent cation such as K⁺.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.     

Effect of adsorbed cations on phosphorus uptake by excised roots

Pretreatment of excised roots of Hordeum vulgare, Zea mays, and Glycine max with various salt solutions affected their subsequent rate of phosphorus absorption from 2 × 10⁻⁵m KH₂PO₄. The rate of absorption was greatest for roots pretreated with trivalent cations, intermediate with divalent cations and lowest with monovalent cations. It appeared that the pretreatment involved a rapid exchange reaction at the root surface which was reversible. A 1 min pretreatment was effective for more than 20 min. The acceleration of phosphorus uptake by roots produced by polyvalent cations may be due partly or entirely to a greater reduction in the electrical potential at the root surface or within the pores of the negatively charged cell wall by polyvalent cations than by monovalent cations.     

Adenosine triphosphatase from soybean callus and root cells

The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺ > Sr²⁺) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K⁺ > Rb⁺ > Choline⁺ > Na⁺ > Li⁺ > NH₄⁺ > Cs⁺ > tris⁺. Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na⁺ over K⁺. No synergism between K⁺ and Na⁺ was found with preparations from either cell source.     

Co-transport of anions and neutral solutes with cations across charged biological membranes. Effects of surrface potential on uptake kinetics

General rate equations have been developed for the co-transport of an anion with one or two cations across a negatively charged biological membrane. The effects of surface potential on the kinetical parameters of co-transport of monovalent anions with monovalent cations have been investigated in more detail. The influence of changes in the surface potential on ion uptake kinetics appears to be markedly affected by the properties of the co-transport system. This can be shown by investigating boundary cases of the general model, namely (a) random order of binding of the ions, (b) anion binds before cations, (c) cations bind before anion. Since the effects of the surface potential are different for these three cases, these effects might serve as (additional) discrimination criteria.The effect of the surface potential on anion uptake kinetics via a co-transport system to which two cations can bind is rather complex: maxima or minima of the apparent affinity constant K_m of anion uptake may occur. Not only the magnitude of the effect of changes in the surface potential, but also its direction (stimulation, inhibition), is influenced by the co-substrate (cation) concentration. Such effects may also occur if only one cation can bind to the translocator, provided that OH^? ions compete for the anion transport site.In addition, the case of co-transport of a neutral solute with a monovalent cation has been investigated. It has been shown, that monovalent cation has been investigated. It has been shown, that also in this case, the effect of changes in the surfaces potential is affected by the order of binding of the substrates to the translocator.     

Differences in the interaction of inorganic and organic (hydrophobic) cations with phosphatidylserine membranes

The interaction of phosphatidylserine dispersions with “hydrophobic”, organic cations (acetylcholine, tetraethylammonium ion) is compared with that of simple inorganic cations (Na⁺, Ca²⁺); differences in the hydration properties of the two classes of ions exist in the bulk phase as evident from spin-lattice relaxation time T₁, measurements. It is shown that the reaction products (cation-phospholipid) differ markedly in their physicochemical behaviour. With increasing concentration both classes of ions reduce the ζ-potential of phosphatidylserine surfaces, the monovalent inorganic cations being only slightly more effective than the hydrophobic cations. Inorganic cations cause precipitation of the lipid once the surface charge of the bilayer is reduced to a certain threshold value. This is not the case with the organic cations. The difference is probably associated with the different hydration properties of the resulting complexes. Thus binding of Ca²⁺ causes displacement of water of hydration and formation of an anhydrous, hydrophobic calcium-phosphatidylserine complex which is insoluble in water, whereas the product of binding of the organic cations is hydrated, hydrophilic and water soluble. The above findings are consistent with NMR results which show that the phosphodiester group is involved in the binding of both classes of cations as well as being the site of the primary hydration shell. Besides affecting interbilayer membrane interactions such as those involved in cell adhesion and membrane fusion, the binding of both classes of cation can affect the molecular packing within a bilayer.     

The effect of metal ions on the amidolytic activity of human factor Xa (Activated Stuart-Prower Factor)

The effect of metal ions on human activated Factor X (Factor X_a) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca²⁺, Mn²⁺, and Mg²⁺ enhanced Factor X_a amidolytic activity with K_m values of 30 μm, 20 μm, and 1.4 mm, respectively. Na⁺ activation of Factor X_a amidolytic activity was also found. The K_m for Na⁺ activation was 0.31 m. Both the divalent metal ions and Na⁺ increased the affinity of Factor X_a for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor X_a. However, K⁺ was a competitive inhibitor of the Na⁺ activation (K_i = 0.14 m). Lanthanide ions inhibited Factor X_a amidolytic activity. Gd³⁺ inhibition of Factor X_a hydrolysis of S2222 was noncompetitive and had a K_i of 3 μm. The lanthanide ion inhibition could not be reversed by Ca²⁺ even when Ca²⁺ was present in a 1000-fold excess over its K_m indicating nonidentity of the Factor X_a lanthanide and Ca²⁺ binding sites. It is concluded that the Factor X_a Ca²⁺ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg²⁺, Na⁺, and K⁺ may be physiological regulators of Factor X_a activity.     

Effects of inorganic solutes on the binding of auxin

The binding of α-naphthaleneacetic acid (¹⁴C-NAA) to pelletable particulates from corn (Zea mays) coleoptiles was found to be influenced by inorganic solutes. La³⁺, Ca²⁺, and Mg²⁺ increased the binding whereas monovalent cations did not. The concentrations of CaCl₂ which increased auxin binding were similar to those which inhibited coleoptile elongation in the presence of auxin. These results are interpreted as suggesting that the alteration of hormonal effectiveness by some inorganic solutes involves alterations in the attachment of the hormone to binding sites in the cell.     

Diffusion NMR-based comparison of electrostatic influences of DNA on various monovalent cations

Counterions are important constituents for the structure and function of nucleic acids. Using ⁷Li and ¹³³Cs nuclear magnetic resonance (NMR) spectroscopy, we investigated how ionic radii affect the behavior of counterions around DNA through diffusion measurements of Li⁺ and Cs⁺ ions around a 15-bp DNA duplex. Together with our previous data on ²³Na⁺ and ¹⁵NH₄⁺ ions around the same DNA under the same conditions, we were able to compare the dynamics of four different monovalent ions around DNA. From the apparent diffusion coefficients at varied concentrations of DNA, we determined the diffusion coefficients of these cations inside and outside the ion atmosphere around DNA (D_b and D_f, respectively). We also analyzed ionic competition with K⁺ ions for the ion atmosphere and assessed the relative affinities of these cations for DNA. Interestingly, all cations (i.e., Li⁺, Na⁺, NH₄⁺, and Cs⁺) analyzed by diffusion NMR spectroscopy exhibited nearly identical D_b/D_f ratios despite the differences in their ionic radii, relative affinities, and diffusion coefficients. These results, along with the theoretical relationship between diffusion and entropy, suggest that the entropy change due to the release of counterions from the ion atmosphere around DNA is also similar regardless of the monovalent ion types. These findings and the experimental diffusion data on the monovalent ions are useful for examination of computational models for electrostatic interactions or ion solvation.     

The hamster adipocyte adenylate cyclase system II. Regulation of enzyme stimulation and inhibition by monovalent cations

In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na⁺>Li⁺>K⁺>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na⁺>Li⁺>K⁺>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E₁ on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E₁, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.     

Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma

Ca²⁺ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10^?5 and 1.2 · 10^?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca²⁺ binding were studied. All cations tested inhibited Ca²⁺ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na⁺ > K⁺ > Li⁺ ? Cs⁺ and for the divalent and trivalent ions: La³⁺ ? Mn²⁺ > Sr²⁺ ? Ba²⁺ > Mg²⁺. 1 · 10^?3 M caffeine and 1 · 10^?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10^?7 M verapamil, acidosis (pH 6.4), 1^?10^?5 M Mn²⁺ and 1 · 10^?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.     

EFFECTS OF CA AND SR ON ZEA MAYS SEEDLING PRIMARY ROOT GROWTH

Either Ca or Sr in a mineral nutrient medium prevented toxic effects of other nutrient ions on aerated primary roots of maize; other monovalent or divalent, cations substituted for Ca did not. Calcium in the complete nutrient solution, or as CaCl₂ alone, stimulated the division of apical meristem cells in these roots, as compared to division in water alone, but Sr did not. However, the replacement of Ca in the nutrient medium by Sr resulted in the development of additional secondary roots, probably in part by decreasing the rate of cell division in the apical meristem of the primary root and thereby diminishing apical dominance.     

The calculation of partial specific volumes of proteins in guanidine hydrochloride

The effects of various modifiers on the ATPase activity of bovine platelet actomyosin has been studied. The order of activation by monovalent cations was NH₄⁺? K⁺ > Li⁺ > Na⁺. The order of activation by divalent cations was Ca²⁺ > Mn²⁺ = Sr²⁺ > Ba²⁺> Co²⁺ > Mg²⁺ > Zn²⁺. Ethylenediaminetetraacetate inhibits. Activity increased with increasing concentrations of monovalent cations, except for inhibition by increasing concentrations of NH₄⁺ in the presence of Ca²⁺. Adenosine triphosphatase activity was increased by low concentrations of urea and trypsin, but was unaffected by low concentrations of N-ethylmaleimide. For all enzymatic properties where direct comparisons are possible, actomyosin from platelets is unlike that from skeletal muscle, but is similar to that from smooth muscle and non-muscle sources.     

The permeation of organic cations through cAMP-gated channels in mammalian olfactory receptor neurons

The permeation of monovalent organic cations through adenosine 3,5-cyclic monophosphate-(cAMP) activated channels was studied by recording macroscopic currents in excised inside-out membrane patches from the dendritic knobs of isolated mammalian olfactory receptor neurons (ORNs). Current-voltage relations were measured when bathing solution Na⁺ was replaced by monovalent organic cations. Permeability ratios relative to Na⁺ ions were calculated from changes in reversal potentials. Some of the small organic cations tested included ammonium (NH ₄ ⁺ ), hydroxylammonium and formamidinium, with relative permeability ratios of 1.41, 2.3 and 1.01 respectively. The larger methylated and ethylated ammonium ions studied included: DMA (dimethylammonium), TMA (tetramethylammonium) and TEA (tetraethylammonium) and they all had permeability ratios larger than 0.09. Even large cations such as choline, arginine and tris(hydroxymethyl)aminomethane (Tris) were appreciably permeant through the cAMP-activated channel with permeability ratios ranging from 0.19 to 0.7. The size of the permeating cations, as assessed by molecular weight, was a good predictor of the permeability. The permeability sequence of the cAMP-activated channel in our study was P_NH4 > P_Na > p_DMA > p_TMA > P_Choline > P_TEA. Higher permeability ratios of hydroxylammonium, arginine and tris(hydroxymethyl)aminomethane cannot be explained by ionic size alone. Our results indicate that: (i) cAMP-activated channels poorly select between monovalent cations; (ii) the pore dimension must be at least 6.5 × 6.5 Å, in order to allow TEA and Tris to permeate and (iii) molecular sieving must be an important mechanism for the permeation of large organic ions through the channels with specific ion binding playing a smaller role than in other structurally similar channels. In addition, the results clearly indicate that cyclic nucleotide-gated (CNG) channels in different cells are not the same, the olfactory CNG channel being different from that of the photoreceptors, particularly with respect to the permeation of large organic cations, which the ORN channels allow to permeate readily.This work was supported by the Australian Research Council of Australia.     

Equilibrium and kinetic study of sodium- and potassium-induced conformational changes of apo-alpha-lactalbumin

Equilibrium and kinetics of Na+-and K+-induced conformational changes of apo-alpha-lactalbumin were studied by means of circular dichroism. While apo-alpha-lactalbumin was considerably unfolded in the absence of Na+ or K+ in 20 mM Tris at pH 8.0 and 25 degrees, both the monovalent cations restored the tertiary structure of the protein. Apparent binding constants of Na+ and K+ to the apoprotein were estimated from the equilibria of the Na+- and K+-induced conformational changes. Based on kinetic data of the conformational changes induced by the monovalent cations, binding mechanism of the ions to the apo-protein was examined. Bound alkali-metal ions stabilize the native-like state and an activated state in the unfolding-refolding reaction of the apoprotein.     

Gas chromatographic measurement of urinary 5-fluoro-2'-deoxyuridine levels after barium salt precipitation and sephadex LH-20 cleanup

A fluorescent photoaffinity label—8-azido-1-N⁶-etheno-adenosine 5′-triphosphate (8-N₃ε ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F₁ATPase from Micrococcus luteus. 8-N₃ε ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F₁ATPase in the presence of the label and Mg²⁺ ions inhibits the enzyme irreversibly. The fluorescent label is bound preferentially to the β subunit of the enzyme. Labeling and inactivation are decreased by protection with ATP or ADP.     

Molecular dynamics and simulation analysis against superoxide dismutase (SOD) target of Micrococcus luteus with secondary metabolites from Bacillus licheniformis recognized by genome mining approach

Micrococcus luteus, also known as M. luteus, is a bacterium that inhabits mucous membranes, human skin, and various environmental sources. It is commonly linked to infections, especially among individuals who have compromised immune systems. M. luteus is capable of synthesizing the enzyme superoxide dismutase (SOD) as a component of its protective response to reactive oxygen species (ROS). This enzyme serves as a promising target for drug development in various diseases. The current study utilized a subtractive genomics approach to identify potential therapeutic targets from M. luteus. Additionally, genome mining was employed to identify and characterize the biosynthetic gene clusters (BGCs) responsible for the production of secondary metabolites in Bacillus licheniformis (B. licheniformis), a bacterium known for its production of therapeutically relevant secondary metabolites. Subtractive genomics resulted in identification of important extracellular protein SOD as a drug target that plays a crucial role in shielding cells from damage caused by ROS. Genome mining resulted in identification of five potential ligands (secondary metabolites) from B. licheniformis such as, Bacillibactin (BAC), Paenibactin (PAE), Fengycin (FEN), Surfactin (SUR) and Lichenysin (LIC). Molecular docking was used to predict and analyze the binding interactions between these five ligands and target protein SOD. The resulting protein–ligand complexes were further analyzed for their motions and interactions of atoms and molecules over 250 ns using molecular dynamics (MD) simulation analysis. The analysis of MD simulations suggests, Bacillibactin as the probable candidate to arrest the activities of SOD. All the five compounds reported in this study were found to act by directly/indirectly interacting with ROS molecules, such as superoxide radicals (O₂–) and hydrogen peroxide (H₂O₂), and transforming them into less reactive species. This antioxidant activity contributes to its protective effects against oxidative stress-induced damage in cells making them likely candidate for various applications, including in the development of antioxidant-based therapies, nutraceuticals, and functional foods.