Development of an electrostatic model predicting copper toxicity to plants

The focus of the present study was to investigate the mechanisms for the alleviation of Cu toxicity in plants by coexistent cations (e.g. Al(3+), Mn(2+), Ca(2+), Mg(2+), H(+), Na(+), and K(+)) and the development of an electrostatic model to predict 50% effect activities (EA50s) accurately. The alleviation of Cu(2+) toxicity was evaluated in several plants in terms of (i) the electrical potential at the outer surface of the plasma membrane (PM) (Ψ(0)(°)) and (ii) competition between cations for sites at the PM involved in the uptake or toxicity of Cu(2+), the latter of which is invoked by the Biotic Ligand Model (BLM) as the sole explanation for the alleviation of toxicity. The addition of coexistent cations into the bulk-phase medium reduces the negativity of Ψ(0)(°) and hence decreases the activity of Cu(2+) at the PM surface. Our analyses suggest that the alleviation of toxicity results primarily from electrostatic effects (i.e. changes in both the Cu(2+) activity at the PM surface and the electrical driving force across the PM), and that BLM-type competitive effects may be of lesser importance in plants. Although this does not exclude the possibility of competition, the data highlight the importance of electrostatic effects. An electrostatic model was developed to predict Cu(2+) toxicity thresholds (EA50s), and the quality of its predictive capacity suggests its potential utility in risk assessment of copper in natural waters and soils.     

Liquid crystalline phase behavior of high molecular weight DNA: a comparative study of the influence of metal ions of different size, charge and binding mode

The ability of Li(+), Na(+), K(+), Rb(+), Cs(+), Mg(2+), Ca(2+), Sr(2+), Ba(2+), Cu(2+), Cd(2+), Al(3+), V(4+), Hg(2+), Pd(2+), Au(3+), and Pt(4+) to provoke liquid crystalline (LC) phases in high molecular weight DNA was investigated. The alkali and alkaline earth metal ions provoked typical cholesteric/columnar structures, whereas transition metal ions precipitated DNA into solid/translucent gel-like aggregates. Heavy metal ions reduced viscosity of DNA solution, disrupting rigid, rod-like DNA structure necessary for LC textures. Three-layer quantum mechanical-molecular mechanical (QM/MM) studies of Li(+), Na(+), K(+), Mg(2+), and Ca(2+) binding DNA fragment suggested several possible binding modes of these ions to the phosphate groups. The dianion mode of metal binding, involving the phosphate groups of both strands of DNA, allowed for higher DNA binding affinity of the alkaline earth metal ions. These results have implications in understanding the biological role of metal ions and developing DNA-based sensors and nanoelectronic devices.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Mannose-Escherichia coli interaction in the presence of metal cations studied in vitro by colorimetric polydiacetylene/glycolipid liposomes

Supramolecular assemblies of liposomes (vesicles) made of diacetylenic lipids and synthetic mannoside derivative glycolipid receptors were successfully used to mimic the molecular recognition occurring between mannose and Escherichia coli. This specific molecular recognition was translated into visible blue-to-red color transition (biochromism) of the polymerized liposomes, readily quantified by UV-visible spectroscopy. Some transition metal cations (Cd(2+), Ag(+), Cu(2+), Fe(3+), Zn(2+) and Ni(2+)) and alkali earth metal cations (Ca(2+), Mg(2+) and Ba(2+)) were introduced into the system to analyze their effects on specific biochromism. Results showed that the presence of Cd(2+), Ag(+), Ca(2+), Mg(2+) and Ba(2+) enhanced biochromism. A possible enhancement mechanism was proposed in the process of bacterial adhesion to host cells. However, Cu(2+), Fe(3+), Zn(2+) and Ni(2+) exhibited inhibitory effects that cooperated with diacetylene lipid with a carboxylic group and increased the rigidity of the liposomal outer leaflet, blocking changes in the side chain conformation and electrical structure of polydiacetylene polymer during biochromism.     

Kinetic properties of a magnesium ion- and calcium ion-stimulated adenosine triphosphatase from the outer-membrane fraction of rat spleen mitochondria

1. Isolated outer membranes from rat spleen mitochondria can be stored in liquid N(2) for several weeks without significant loss of ATPase (adenosine triphosphatase) activity. 2. The ATPase reaction has a broad pH optimum centering on neutral pH, with little significant activity above pH9.0 or below pH5.5. 3. A sigmoidal response of the ATPase activity to temperature is observed between 0 and 55 degrees C, with complete inactivation at 60 degrees C. The Arrhenius plot shows that the activation energy above the transition temperature (22 degrees C) (E(a)=144kJ/mol) is one-third of that calculated for below the transition temperature (E'(a)=408kJ/mol). 4. The outer-membrane ATPase (K(m) for MgATP=50mum) is inactive unless Mg(2+) is added, whereas the inner-membrane ATPase (K(m) for ATP=11mum) is active without added Mg(2+) unless the mitochondria have been depleted of all endogenous Mg(2+) (by using ionophore A23187). 5. The substrate for the outer-membrane ATPase is a bivalent metal ion-nucleoside triphosphate complex in which Mg(2+) (K(m)=50mum) can be replaced effectively by Ca(2+) (K(m)=6.7mum) or Mn(2+), and ATP by ITP. Cu(2+), Co(2+), Sr(2+), Ba(2+), Ni(2+), Cd(2+) and Zn(2+) support very little ATP hydrolysis. 6. Univalent metal ions (Na(+), K(+), Rb(+), Cs(+) and NH(4) (+), but not Li(+)) stimulate the MgATPase activity (<10%) at low concentrations (50mm), but, except for K(+), are slightly inhibitory (20-30%) at higher concentrations (500mm). 7. The Mg(2+)-stimulated ATPase activity is significantly inhibited by Cu(2+) (K(i)=90mum), Ni(2+) (K(i)=510mum), Zn(2+) (K(i)=680mum) and Co(2+) (K(i)=1020mum), but not by Mg(2+), Ca(2+), Ba(2+) or Sr(2+). 8. The outer-membrane ATPase is insensitive to the inhibitors oligomycin, NN'-dicyclohexylcarbodiimide, NaN(3), ouabain and thiol-specific reagents. A significant inhibition is observed at high concentrations of AgNO(3) (0.5mm) and NaF (10mm). 9. The activity towards MgATP is competitively inhibited by the product MgADP (K(i)=0.7mm) but not by the second product P(i) or by 5'-AMP.     

Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca(2+)-stimulated ATPase (adenosine triphosphatase), a Mg(2+)-stimulated ATPase, and an Na(+)+K(+)-stimulated Mg(2+)-dependent ouabain-sensitive ATPase. 2. The Ca(2+)-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3-4mm-Ca(2+) when 4mm-ATP was present as a substrate, and was not influenced by Na(+), K(+) or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca(2+)-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg(2+), Ca(2+), Mn(2+), Co(2+), Sr(2+), Ba(2+), Zn(2+), Cu(2+).     

Factors contributing to dwarfing in the mangrove Avicennia marina

BACKGROUND AND AIMS: In Richards Bay, South Africa, Avicennia marina frequently exhibits a distinct productivity gradient, with tree height decreasing markedly from 6-10 m in the fringe zone to <1.5 m in the dwarf zone which is 120 m inland at a slightly higher elevation. In this investigation, soil physico-chemical conditions between fringe and dwarf A. marina were compared and the constraints imposed by any differences on mangrove ecophysiology and productivity determined. METHODS: Soil and plant samples were analysed for inorganic ions using spectrophotometry. Gas exchange measurements were taken with an infrared gas analyser and chlorophyll fluorescence with a fluorometer. Xylem psi was determined with a pressure chamber and chlorophyll content with a chlorophyll absorbance meter. RESULTS: In the dwarf site, soil salinity, total cations, electrical conductivity and soil concentrations of Na(+), K(+), Ca(2+), Mg(2+), Zn(2+), Mn(2+) and Cu(2+) were significantly higher than those in the fringe zone. Soil water potential and the concentration of soil P, however, were significantly lower in the dwarf site. In the leaves, Na(+) was the predominant ion and its concentration was 24 % higher in dwarf than fringe mangroves. Leaf concentrations of K(+), Ca(2+), Mg(2+), Mn(2+) and P, however, were significantly lower in dwarf mangroves. Photosynthetic performance, measured by gas exchange and chlorophyll fluorescence, was significantly reduced in the dwarf plants. CONCLUSIONS: The results suggest that hydro-edaphic factors contribute to high soil salinities, low water potentials, water stress and ion imbalance within tissues including P deficiency, which in interaction, contribute to dwarfing in Avicennia marina.     

A scale of metal ion binding strengths correlating with ionic charge, Pauling electronegativity, toxicity, and other physiological effects

Equilibrium constants for binding to plant plasma membranes have been reported for several metal ions, based upon adsorption studies and zeta-potential measurements. LogK values for the ions are these: Al(3+), 4.30; La(3+), 3.34; Cu(2+), 2.60; Ca(2+) and Mg(2+), 1.48; Na(+) and K(+), 0 M(-1). These values correlate well with logK values for ion binding to many organic and inorganic ligands. LogK values for metal ion binding to 12 ligands were normalized and averaged to produce a scale for the binding of 49 ions. The scale correlates well with the values presented above (R(2)=0.998) and with ion binding to cell walls and other biomass. The scale is closely related to the charge (Z) and Pauling electronegativity (PE) of 48 ions (all but Hg(2+)); R(2)=0.969 for the equation (Scale values)=-1.68+Z(1.22+0.444PE). Minimum rhizotoxicity of metal ions appears to be determined by binding strengths: log a(PM,M)=1.60-2.41exp[0.238(Scale values)] determines the value of ion activities at the plasma membrane surface (a(PM,M)) that will ensure inhibition of root elongation. Additional toxicity appears to be related to softness, accounting for the great toxicity of Ag(+), for example. These binding-strength values correlate with additional physiological effects and are suitable for the computation of cell-surface electrical potentials.     

Electrical potentials of plant cell walls in response to the ionic environment

Electrical potentials in cell walls (psi(Wall)) and at plasma membrane surfaces (psi(PM)) are determinants of ion activities in these phases. The psi(PM) plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of psi(Wall). psi(Wall) from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of psi(Wall) with effectiveness in the order Al(3+) > La(3+) > H(+) > Cu(2+) > Ni(2+) > Ca(2+) > Co(2+) > Cd(2+) > Mg(2+) > Zn(2+) > hexamethonium(2+) > Rb(+) > K(+) > Cs(+) > Na(+). This order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of psi(Wall), and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for psi(Wall) were in close agreement, usually, and we consider psi(Wall) to be at least proportional to the actual Donnan potentials. psi(Wall) and psi(PM) display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. psi(Wall) is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion.     

Evaluation of an electrostatic toxicity model for predicting Ni(2+) toxicity to barley root elongation in hydroponic cultures and in soils

Assessing environmental risks of metal contamination in soils is a complex task because the biologically effective concentrations of metals in soils vary widely with soil properties. The factors influencing the toxic effect of nickel (Ni) on root growth of barley (Hordeum vulgare) were re-evaluated using published data from both soil and hydroponic cultures. The electrical potential (ψ(0) (o) ) and ion activities ({I(z) }(0) (o) ) at the outer surfaces of root-cell plasma membranes (PMs) were computed as the basis of the re-evaluation. The reanalyses demonstrated that root growth was related to: the Ni(2+) activity at the PM surface, ({Ni(2+) }(0) (o) ); calcium (Ca) deficiency (related to {Ca(2+) }(0) (o) ); osmotic effects; and modification of intrinsic Ni(2+) toxicity by magnesium (Mg(2+) ; this appeared to exert an intrinsic (specific) ameliorating effect on intrinsic Ni(2+) toxicity). Electrostatic toxicity models (ETM) were developed to relate root growth to these factors (R(2) > 0.751). Based on the ETM developed in soil culture and a Ni(2+) solid-solution partitioning model, critical metal concentrations in soils linked to a biological effect were well predicted for 16 European soils with a wide range of properties, indicating the potential utility of ETM in risk assessment of metals in terrestrial ecosystems.     

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.     

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.     

High resolution structures of p-aminobenzamidine- and benzamidine-VIIa/soluble tissue factor: unpredicted conformation of the 192-193 peptide bond and mapping of Ca2+, Mg2+, Na+, and Zn2+ sites in factor VIIa

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca(2+) ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca(2+) and Mg(2+), and the Ca(2+) sites in FVIIa that could be specifically occupied by Mg(2+) are unknown. Furthermore, FVIIa contains a Na(+) and two Zn(2+) sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca(2+), Mg(2+), Na(+), and Zn(2+). The crystal diffracted to 1.8A resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca(2+) and three bound Mg(2+). The EGF1 domain contains one Ca(2+) site, and the protease domain contains one Ca(2+), one Na(+), and two Zn(2+) sites. (45)Ca(2+) binding in the presence/absence of Mg(2+) to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly(193) in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87A resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.     

Different cation stresses affect specifically osmotic root hydraulic conductance, involving aquaporins, ATPase and xylem loading of ions in Capsicum annuum, L. plants

In order to study the effect of nutrient stress on water uptake in pepper plants (Capsicum annuum L.), the excess or deficiency of the main cations involved in plant nutrition (K(+), Mg(2+), Ca(2+)) and two different degrees of salinity were related to the activity of plasma membrane H(+)-ATPase, the pH of the xylem sap, nutrient flux into the xylem (J(s)) and to a number of parameters related to water relations, such as root hydraulic conductance (L(0)), stomatal conductance (g(s)) and aquaporin activity. Excess of K(+), Ca(+) and NaCl produced a toxic effect on L(0) while Mg(2+) starvation produced a positive effect, which was in agreement with aquaporin functionality, but not with ATPase activity. The xylem pH was altered only by Ca treatments. The results obtained with each treatment could suggest that detection of the quality of the nutrient supply being received by roots can be related to aquaporins functionality, but also that each cation stress triggers specific responses that have to be assessed individually.     

Calculated activity of Mn2+ at the outer surface of the root cell plasma membrane governs Mn nutrition of cowpea seedlings

Manganese (Mn) is an essential micronutrient for plant growth but is often toxic in acid or waterlogged soils. Using cowpea (Vigna unguiculata L. Walp.) grown with 0.05-1500 μM Mn in solution, two short-term (48 h) solution culture experiments examined if the effects of cations (Ca, Mg, Na, Al, or H) on Mn nutrition are related to the root cells' plasma membrane (PM) surface potential, ψ(0)(0). When grown in solutions containing levels of Mn that were toxic, both relative root elongation rate (RRER) and root tissue Mn concentration were more closely related to the activity of Mn(2+) at the outer surface of the PM, {Mn(2+)}(0)(0) (R(2)=0.812 and 0.871) than to its activity in the bulk solution, {Mn(2+)}(b) (R(2)=0.673 and 0.769). This was also evident at lower levels of Mn (0.05-10 μM) relevant to studies investigating Mn as an essential micronutrient (R(2)=0.791 versus 0.590). In addition, changes in the electrical driving force for ion transport across the PM influenced both RRER and the Mn concentration in roots. The {Mn(2+)}(b) causing a 50% reduction in root growth was found to be c. 500 to >1000 μM (depending upon solution composition), whilst the corresponding value was 3300 μM when related to {Mn(2+)}(0)(0). Although specific effects such as competition are not precluded, the data emphasize the importance of non-specific electrostatic effects in the Mn nutrition of cowpea seedlings over a 1×10(5)-fold range of Mn concentration in solution.     

Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity

In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates. In particular, dissociation constant values (K(d)) of 23 and 27 mM were measured for Cs(+) and Ca(2+), respectively, using, as substrate, spermine, a polyamine of plasma. An additional cation-binding area, where metal ions such as Cs(+) (K(d) congruent with 0.1 mM) and Na(+) (K(d) congruent with 54 mM) bind without affecting the enzyme activity, was found by NMR.     

A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.