Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid     

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes II. Extrinsic influence of cations

Use-dependent declines of Na⁺ currents in myelinated frog nerve fibres were measured during a train of depolarizing pulses in solutions containing tetrodotoxin (TTX) or saxitoxin (STX). The following effects of external monovalent (Na⁺), divalent (Ca²⁺, Mg²⁺) and trivalent (La²⁺) cations on use dependence were found: Increasing the Ca²⁺ concentration from 2 to 8 mM shifts its voltage dependence by 20 mV whereas no significant use-dependent decline occurred at 0.2 mM Ca²⁺. Doubling the external Na⁺ concentration in 0.2 mM Ca²⁺ solutions did not initiate phasic block. External Mg²⁺ ions induced a smaller, and La²⁺ ions a larger, use dependence. The time constants of the current decline were 4-fold greater in 1.08 mM La²⁺. The static block of Na⁺ currents by La³⁺ could be directly demonstrated by the relief of block during a train of pulses. The results are qualitatively explained by a toxin binding site at the Na⁺ channel whose affinity for TTX or STX depends oni) the gating conformation of the channel, probably the inactivation andii) the occupancy of a blocking site by di- or trivalent external cations.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Solubilization and purification of the Ni-stimulated arginine-vasopressin binding site of rat brain membranes

Arginine vasopressin binding sites on rat brain membranes were solubilized and purified by affinity chromatography. Membrane protein solubilized with CHAPS bound arginine vasopressin (AVP) only in the presence of divalent cations. Specific binding to the solubilized tissue was maximally stimulated by Ni²⁺, and markedly stimulated by Co²⁺ (30% of maximal binding with Ni²⁺), Zn²⁺ (18%), and Fe²⁺ (11%), parallel to the effects of these ions on the binding of AVP to neural membranes. Binding to solubilized tissue was not stimulated by Mg²⁺, Cu²⁺, Mn²⁺, or Ca²⁺. In the presence of Ni²⁺, binding of AVP to solubilized tissue was reversible, and the dissociation constant (10.5 nM), pH optimum, and time course were virtually identical to those of the membrane-bound AVP binding site. Purification of solubilized AVP-binding proteins by affinity chromatography on AVP-sepharose followed by gel electrophoresis yielded a major band of 55 kdalton molecular weight when purified in the presence of 5 mM Mg²⁺, or a major band of 62 kdaltons when purified in the presence of 1–5 mM Ni²⁺ or 10 M Zn²⁺. By means of a new binding assay involving conjugation of the 62 kdalton fraction to brain membranes, the extent of purification of AVP binding activity was 150-fold in the presence of Ni²⁺. We suggest that the 62 kdalton protein is a component of the Ni-stimulated AVP binding site.     

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn²⁺, Cu²⁺, Mg²⁺ and Ca²⁺ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn²⁺ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn²⁺-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca²⁺/Mg²⁺-binding, Zn²⁺ or Cu²⁺ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca²⁺-binding to HSA synergistically support each other. Zn²⁺ and Cu²⁺ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.     

Investigation of the encapsulation of metal cations (Cu<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript>) by the dipeptide Phe–Phe using natural bond orbital theory and molecular dynamics simulation

Complexes of the dipeptide phenylalanine–phenylalanine (Phe–Phe) with divalent metal cations (Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺) were studied at the B3LYP and MP2 levels of theory with the basis sets 6-311++G(d,p) and 6-31 + G(d) in the gas phase. The relative energies of these complexes indicated that cation–π bidentate/tridentate conformations are more favourable than other conformations with uncoordinated rings. These findings were confirmed by the calculated values of thermodynamic parameters such as the Gibbs free energy. Natural bond orbital (NBO) analysis was carried out to explore the metal–ligand coordination in Phe–Phe–Cu²⁺/Zn²⁺ complexes. Possible orbital transitions, types of orbitals and their occupancies were determined for a range of Phe–Phe–Cu²⁺/Zn²⁺ complexes. The charge transfer involved in various orbital transitions was explored by considering the second-order perturbation energy. NBO analysis revealed that the change transfer is stronger when the metal cation uses both the 4s + 4p subshells rather than just its 4p subshell. We also performed molecular dynamics (MD) simulations to check the stability and consistency of the most favourable binding motifs of Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺ with Phe–Phe over time. The structures of the Phe–Phe–Cu²⁺/Zn²⁺/Ca²⁺/Ba²⁺ complexes obtained using MD simulation were found to be in good agreement with those obtained in the DFT-based calculations.

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Graphical Abstract Conformational search on encapsulation of divalent metal cations (Ca²⁺, Zn²⁺, Ca²⁺, Ba²⁺) by the Phe-Phe dipeptide

     

Zinc Absorption by Wheat Seedlings and the Nature of its Inhibition by Alkaline Earth Cations

The effects and interactions of the alkaline earth cations onZn²⁺ absorption were studied in. short-term experiments. Atlow concentrations of Zn²⁺ ( 2 µM), rates of Zn²⁺ absorptionwere linear even in the absence of Ca²⁺ or of other cations.At higher Zn²⁺ concentrations (5 and 10µm), rates werenot linear in the absence of other cations but became linearon addition of 250 µM or more of Ca²⁺, Mg²⁺, Sr²⁺, orBa²⁺. From 0.1 to 10µM Zn²⁺, all alkaline earth cations inhibitedabsorption in the order Mg²⁺ > Ba²⁺ Sr²⁺ = Ca²⁺. Increasingconcentrations of Ca²⁺ or of Mg²⁺ from 0 to 40 mM progressivelydepressed absorption from 1µM ZnCl₂. Increasing Ca²⁺ orMg²⁺ from 40 to 100 mM had no further effect on absorption.Over both high and low ranges of Ca²⁺ or Mg²⁺ concentrations,the affinity of plant roots for Zn²⁺ and the responses of Zn²⁺absorption to temperature, H⁺, and Cu²⁺ were identical. At equalconcentrations over the whole concentration range, Mg²⁺ was30 per cent more effective than Ca²⁺ in inhibiting absorption.At concentrations below 40 mM, Ca²⁺ and Mg²⁺ competed with eachother in their inhibiting effects. At concentrations above 40mM, Ca²⁺ alleviated the extra inhibitory effects of Mg²⁺ insome unknown way. The alkaline earth cations inhibited Zn²⁺ absorption non-competitively.They depressed it to values which would limit vigorous plantgrowth. It is postulated that their effects are important inthe zinc nutrition of plants in soil and in solution cultures.     

An increase of calcium/manganese binding sites in cell ghosts associated with the acquisition of aggregative competence in Dictyostelium discoideum.

In Dictyostelium discoideum, the formation of multicellular aggregates represents the first morphogenetic event that leads ultimately to the construction of fruiting bodies. The altered adhesive properties of the cells can be demonstrated in ghosts derived from them which consist of largely intact membranes containing a few empty vesicles and exploded mitochondria but lacking nuclei, RNA, soluble cytoplasm and ATP [4]. A cofactor requirement for the aggregation of the ghosts can be satisfied by the following divalent cations: Ca²⁺, Mn²⁺, Zn²⁺ and Cu²⁺. In this paper it is shown that associated with the acquisition of aggregative competence is a 15–20-fold increase in the ghosts of sites capable of binding either Ca²⁺ or Mn²⁺ with relatively high affinity.     

Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

Summary 1. We examined the actions of mercury (Hg²⁺) and zinc (Zn²⁺) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique.2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of –80 to 0 mV (mainly L- and N-currents) were reduced by Hg²⁺ and Zn²⁺. The threshold concentration for Hg²⁺ effects was 0.1 µM and that for Zn²⁺ was 10µM. Voltage-activated calcium channel currents were abolished (>80%) with 5µM Hg²⁺ or 200µM Zn²⁺. The peak calcium current was reduced to 50% (IC₅₀) by 1.1µM Hg²⁺ or 69µM Zn²⁺. While Zn²⁺ was much more effective in reducing the T-type calcium channel current—activated by jumping from –80 to –35 mV—Hg²⁺ showed some increased effectiveness in reducing this current.3. The effects of both cations occurred rapidly and a steady state was reached within 1–3 min. While the action of Zn²⁺ was not dependent on an open channel state, Hg²⁺ effects depended partially on channel activation.4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg²⁺ and with higher concentrations (>100µM) of Zn²⁺.5. As Zn²⁺ in the concentration range used had no influence on resting membrane currents, Hg²⁺ caused a clear inward current at concentrations µM.6. In the present study we discuss whether the actions of both metals on voltage-activated calcium channel currents are mediated through the same binding site and how they may be related to their neurotoxic effects.     

The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping

S100B is a homodimeric zinc-, copper-, and calcium-binding protein of the family of EF-hand S100 proteins. Zn²⁺ binding to S100B increases its affinity towards Ca²⁺ as well as towards target peptides and proteins. Cu²⁺ and Zn²⁺ bind presumably to the same site in S100B. We determined the structures of human Zn²⁺- and Ca²⁺-loaded S100B at pH 6.5, pH 9, and pH 10 by X-ray crystallography at 1.5, 1.4, and 1.65 Å resolution, respectively. Two Zn²⁺ ions are coordinated tetrahedrally at the dimer interface by His and Glu residues from both subunits. The crystal structures revealed that ligand swapping occurs for one of the four ligands in the Zn²⁺-binding sites. Whereas at pH 9, the Zn²⁺ ions are coordinated by His15, His25, His 85′, and His 90′, at pH 6.5 and pH 10, His90′ is replaced by Glu89′. The results document that the Zn²⁺-binding sites are flexible to accommodate other metal ions such as Cu²⁺. Moreover, we characterized the structural changes upon Zn²⁺ binding, which might lead to increased affinity towards Ca²⁺ as well as towards target proteins. We observed that in Zn²⁺-Ca²⁺-loaded S100B the C-termini of helix IV adopt a distinct conformation. Zn²⁺ binding induces a repositioning of residues Phe87 and Phe88, which are involved in target protein binding. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Biosorption of metal ions byAzotobacter vinelandii

Azotobacter vinelandii was better than eitherDerxia gummosa orRhizobium trifolii for sorption of UO ₂ ²⁺ . Its maximum binding capacity was 0.25 mmol UO ₂ ²⁺ /g dry biomass with an affinity constant of 333 l/mmol at pH 4.1 according to the Langmuir model. In a semisynthetic medium,A. vinelandii showed the highest sorption capacity in the early stationary phase. The binding of UO ₂ ²⁺ , Cu²⁺, Ca²⁺ and Zn²⁺ was affected by the pH of the solution. With HCl as eluent, virtually all the sorbed UO ₂ ²⁺ was released. The presence of Cu²⁺, Cd²⁺, Ca²⁺, and Zn²⁺ inhibited the UO ₂ ²⁺ biosorption whereas Mg²⁺ and K⁺ had no effect.     

Metals are directly involved in the redox interconversion of Saccharomyces cerevisiae glutathione reductase

Summary Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 µM EDTA or 10 µM DETAPAC yielding full protection. Ag⁺, Zn²⁺ and Cd²⁺ potentiated the redox inactivation promoted by NADPH alone, while Cr³⁺, Fe²⁺, Fe³⁺, Cu⁺, and Cu²⁺ protected the enzyme. The Zn²⁺ and Cd²⁺ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn²⁺ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn²⁺ or Cd²⁺.The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP⁺ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.Abbreviations DETAPAC diethylenetriaminepentaacetic acid - 2,5-ADP-Sepharose-N⁶-(6-aminohexyl) adenosine 2,5-bisphosphateSepharose     

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

Summary Leakage of ions (Na⁺, K⁺) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by pore-forming agent has been studied. Leakage from intact cells isinduced by protons and by divalent cations such as Cu²⁺, Cd²⁺ or Zn²⁺. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented by low concentrations of the same cations and by higher concentrations of Ca²⁺, Mn²⁺ or Ba²⁺; Mg²⁺, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca²⁺) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na⁺, K⁺, Rb⁺, NH ₄ ⁺ ) and divalent (Mg²⁺, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn²⁺ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.     

Mechanism of luminal Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca²⁺-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channels closed conformations. Vacuolar Ca²⁺ and Mg²⁺ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a permeable blocker. Vacuolar Ca²⁺—with a much higher affinity than Mg²⁺—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca²⁺-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channels closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca²⁺ at the vacuolar side. The specificity for Ca²⁺ compared to Mg²⁺ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca²⁺ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.Abbreviation SV Slow vacuolar     

Association of aureolic acid antibiotic, chromomycin A3 with Cu2+ and its negative effect upon DNA binding property of the antibiotic

Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu²⁺. CHR forms a high affinity 2:1 (CHR:Cu²⁺) complex with dissociation constant of 0.08 × 10⁻¹⁰ M² at 25°C, pH 8.0. The affinity of CHR for Cu²⁺ is higher than those for Mg²⁺ and Zn²⁺ reported earlier from our laboratory. CHR binds preferentially to Cu²⁺ in presence of equimolar amount of Zn²⁺. Complex formation between CHR and Cu²⁺ is an entropy driven endothermic process. Difference between calorimetric and van’t Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)₂:Cu²⁺] complex assumes a structure different from either of the Mg²⁺ and Zn²⁺ complex reported earlier. Both [(CHR)₂:Mg²⁺] and [(CHR)₂:Zn²⁺] complexes are known to bind DNA. In contrast, [(CHR)₂:Cu²⁺] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5′-CCGGCGCCGG-3′). In order to interact with double helical DNA, the (antibiotic)₂ : metal (Mg²⁺ and Zn²⁺) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu²⁺ complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg²⁺and Zn²⁺. The results also indicate that CHR has a potential for chelation therapy in Cu²⁺ accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.     

Use of Soil Amendments to Reduce Nitrogen,Phosphorus and Heavy Metal Availability

The primary objectives of this study were to determine (1) the exchange characteristics of various soil amendments using a range of salt solutions, (2) the effect of selected soil amendments on heavy metal (Cu²⁺, Pb²⁺, and Zn²⁺) availability, and (3) the effect of selected soil amendments on NH₄ ⁺ and P availability. The CEC of zeolite and red mud obtained using solutions of 0.1?M BaCl₂ and 0.1?M BaCl₂/NH₄Cl were significantly lower than values obtained using 1?M KCl and 1?M NH₄Cl. The higher CEC obtained with monovalent cations indicated that larger divalent cations could not enter the mineralogical framework of zeolite and red mud, and, consequently, a number of exchange sites were only accessible to the smaller monovalent cations. These findings suggest that 1?M KCl and 1?M NH4NO3 should be used as the extracting solutions to obtain the best estimation of CEC and ECEC of red mud and zeolite. The ability of red mud, zeolite, and calcium phosphate (Ca-P), mixed at rates of 0%, 5%, 10%, and 20% (w/w), to sorb Cu²⁺, Pb²⁺, and Zn²⁺ generally followed the order: red mud>zeolite>>Ca-P, while the affinity sequence for these metals followed the order: Pb²⁺≥Cu²⁺>>Zn²⁺. The higher affinity of the sand/amendment mixtures for Pb²⁺ and Cu²⁺ relative to Zn²⁺ was attributed to metal hydrolysis and subsequent specific adsorption as Pb(OH)⁺ and Cu(OH)⁺. Zinc was considered to have been primarily sorbed as the divalent cation species. Rates of 5% (w/w) adequately reduced the availability of heavy metals to concentrations below environmental guidelines based on the Toxicity Characteristic Leaching Procedure. Red mud and zeolite added at a rate of 10% (w/w) to the A and B horizon of a sandy soil significantly increased their ability to remove NH₄ ⁺ from solution, but had negligible effect on P sorption compared with unamended soils. Increased NH₄ ⁺ removal was attributed to the associated increase in CEC and the greater selectivity of the exchange sites for this cation relative to resident exchangeable Ca²⁺ and Na⁺. The absence of P sorption by these two amendments was attributed to the high pH and predominantly negative surface charge of the red mud and the lack of sorption sites in zeolite. Gypsum, on the other hand, tended to depress NH₄ ⁺ retention but markedly increased P sorption. The depressive effect on NH₄ ⁺ was due to increased competition between NH₄ ⁺ and Ca₂ ⁺ for a limited number of exchange sites, while formation of calcium phosphates of low solubility was the possible mechanism for increased P sorption.     

Multivalent cations and ligand affinity of the type 1 insulin-like growth factor receptor on P2A2-LISN muscle cells

Mouse P₂A₂-LISN myoblasts are transfected cells that overexpress the human type 1 insulin-like growth factor (IGF) receptor. Because the type 1 IGF receptor is the major binding site for both IGF-I and IGF-II, this cell line is an excellent model to determine the effect of multivalent cations on ligand binding specifically to this type of receptor. Competitive binding assays were performed to characterize IGF binding and Scatchard analysis to quantify affinity (K_a). ¹²⁵I-IGF-I, ¹²⁵I-IGF-II, and ¹²⁵I-R³-IGF-I bind only to the type 1 IGF receptor on these cells. Zn²⁺ increased binding of the three ligands to the type 1 IGF receptor by 17 to 35%. Cd²⁺ significantly increased binding of ¹²⁵I-IGF-I, although by only 8%. La³⁺ and Cr³⁺ did not effect binding. Au³⁺ decreased IGF binding by approximately 56%. Scatchard analysis produced nonlinear concave-down plots yielding binding constants for high and low affinity sites. Zn²⁺ increased the strength of only the high affinity sites. Au³⁺ decreased the affinity of both high and low affinity sites. Zn²⁺ increased binding with a half-maximal effect between 40 μM and 60 μM. Half-maximal dose of Au³⁺ was > 130 μM. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) and only these cations were found to affect receptor binding indicating similar mechanisms of action. Thus, multivalent cations may alter IGF binding to cell surface receptors ultimately controlling growth. Physiologically this may be especially important for the growth promoting effects of Zn²⁺. J. Cell. Physiol. 176:392–401, 1998. © 1998 Wiley-Liss, Inc.     

Binding and Electrostatic Attraction of Lanthanum (La3+) and Aluminum (Al3+) to Wheat Root Plasma Membranes

A general model for the sorption of trivalent cations to wheat-root (Triticum aestivum L cv. Scout 66) plasma membranes (PM) has been developed and includes the first published coefficients for La³⁺ and Al³⁺ binding to a biological membrane. Both ions are rhizotoxic, and the latter ion is the principal contributor to the toxicity of acidic soils around the world. The model takes into account both the electrostatic attraction and the binding of cations to the negatively charged PM surface. Ion binding is modeled as the reaction P ⁻+I ^Z⇌PI ^{Z −1} in which P ⁻ represents a negatively charged PM ligand, located in an estimated area of 540 ?², and I ^Z represents an ion of charge Z. Binding constants for the reaction were assigned for K⁺ (1 m ⁻¹) and Ca²⁺ (30 m ⁻¹) and evaluated experimentally for La³⁺ (2200 m ⁻¹) and H⁺ (21,500 m ⁻¹). Al sorption is complicated by Al³⁺ hydrolysis that yields hydroxoaluminum species that are also sorbed. Binding constants of 30 and 1 m ⁻¹ were assigned for AlOH²⁺ and Al(OH)⁺ ₂, respectively, then a constant for Al³⁺ (20,000 m ⁻¹) was evaluated experimentally using the previously obtained values for K⁺, Ca²⁺ and H⁺ binding. Electrostatic attraction was modeled according to Gouy-Chapman theory. Evaluation of parameters was based upon the sorption of ions to PM vesicles suspended in solutions containing variable concentrations of H⁺, Ca²⁺ and La³⁺ or Al³⁺. Use of small volumes, and improved assay techniques, allowed the measurement of concentration depletions caused by sorption to vesicles. Some independent confirmation of our model is provided by substantial agreement between our computations and two published reports of La³⁺ effects upon zeta potentials of plant protoplasts. The single published report concerning the electrostatic effects of Al on cell membranes is in essential agreement with the model. Received: 6 January 1997/Revised: 6 June 1997