Binding of Cd2+, Ni2+, Cu2+ and Zn2+ by isolated envelopes of Pseudomonas fluorescens

Abstract Cell envelopes of Pseudomonas fluorescens , cytoplasmic membrane, peptidoglycan and outer membrane were obtained from a fractionation procedure and tested for their metal binding capacity. Isolated envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) were chemically modified and functional carboxyl groups transformed to electropositive amine groups, using carbodiimide ethylenediamine. Transformation of carboxyl groups was evaluated by measuring total amine groups in all fractions (modified or not). Using equilibrium dialysis and Scatchard plots for the data, we have established that isolated unmodified cell envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) possess at least two types of metal binding sites with different association constants ( K _a and K '_a). Introduction of positive charges into the bacterial envelopes resulted in the disappearance of one type of metal binding site which had the highest association constant value for Ni²⁺, Cu²⁺ and Zn²⁺. All fractions, modified or not, always presented at least two types of binding sites with different association constants for Cd²⁺.     

Hydrolysis of glucuronide-based substrates mediated by tungsten, Cu2+, Fe2+, and Zn2+

A variety of metal microprojectiles are currently used for carrying foreign DNA into living cells via particle-acceleration techniques. While developing a microprojectile-mediated protocol for transforming cells of sugarbeet ( Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-gluc) in the absence of gusA DNA encoding β-D-glucuronidase (GUS). Tungsten microcarriers, but not gold or silicon carbide, proved capable of catalyzing the cleavage of the glucuronide residue from three histochemical substrates evaluated: X-gluc, salmon X-gluc and magenta X-gluc. Indigo-stained sugarbeet cells were observed following bombardment with tungsten in the absence of DNA. Addition of oxidative catalysts to tungsten microcarriers during substrate incubation had no apparent effect on the metal-mediated catalysis. Treatment of microcarriers with Proteinase K and heat ruled out the presence of enzymes. Microbiological evaluation indicated the absence of contaminating microbes. Similarly, metal-catalyzed hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronic acid (4-MUG) was observed in the presence of tungsten spheres but not with gold or silicon carbide particles. With this substrate, hydrolysis also occurred with millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions. Consequently, careful monitoring of DNA-minus controls and avoidance of millimolar concentrations of Cu²⁺, Fe²⁺ and Zn²⁺ ions are recommended in microprojectile bombardment experiments where transient assays for gusA expression are performed.     

The regulation of Mn2+ and Cu2+ uptake in cells of the yeast Candida utilis grown in continuous culture

Abstract Transport of Mn²⁺ was repressed in Candida utilis cells grown in continuous culture in high-Mn²⁺ (100 μM Mn²⁺) medium as compared to cells grown in basic (0.45 μM Mn²⁺) and low-Mn²⁺ (< 0.05 μM Mn²⁺) media. In contrast, no repression of Cu²⁺ uptake occurred in high-Cu²⁺-grown (25 μM Cu²⁺) cells as compared to cells grown in basic medium (0.54 μM Cu²⁺). Cu²⁺-limited cells did not hyperaccumulate Cu²⁺ and there was not significant difference in initial uptake rates for all 3 Cu²⁺ conditions. Mn²⁺ uptake appears to be regulated by a mechanism sensitive to the external Mn²⁺ concentration, whereas Cu²⁺ transport is not governed in this way by the external Cu²⁺.     

Effects of interrupted K+ supply on growth and uptake of K+, Ca2+, Mg2+ and Na+ in spring wheat

Effects of interrupted K⁺ supply on different parameters of growth and mineral cation nutrition were evaluated for spring wheat (Triticum aestivum L. cv. Svenno). K⁺ (2.0 mM) was supplied to the plants during different periods in an otherwise complete nutrient solution. Shoot growth was reduced before root growth after interruption in K⁺ supply. Root structure was greatly affected by the length of the period in K⁺ -free nutrient solution. Root length was minimal, and root branching was maximal within a narrow range of K⁺ status of the roots. This range corresponded to cultivation for the last 1 to 3 days, of 11 in total, in K⁺ -free nutrient solution, or to continuous cultivation in solution containing 0.5 to 2 mM K⁺. In comparison, both higher and lower internal/external K⁺ concentrations had inhibitory effects on root branching. However, the differing root morphology probably had no significant influence on the magnitude of Ca²⁺, Mg²⁺ and Na⁺ uptake. Uptake of Ca²⁺ and especially Mg²⁺ significantly increased after K⁺ interruption, while Na⁺ uptake was constant in the roots and slowly increased in the shoots. The two divalent cations could replace K⁺ in the cells and maintain electroneutrality down to a certain minimal range of K⁺ concentrations. This range was significantly higher in the shoot [110 to 140 μmol (g fresh weight)^?1] than in the root [20 to 30 μmol (g fresh weight)^?1]. It is suggested that the critical K⁺ values are a measure of the minimal amount of K⁺ that must be present for physiological activity in the cells. At the critical levels, K⁺ (⁸⁶Rb) influx and Ca²⁺ and Mg²⁺ concentrations were maximal. Below the critical K⁺ values, growth was reduced, and Ca²⁺ and Mg²⁺ could no longer substitute for K⁺ for electrostatic balance. In a short-term experiment, the ability of Ca²⁺ to compete with K⁺ in maintaining electroneutrality in the cells was studied in wheat seedlings with different K⁺ status. The results indicate that K⁺, which was taken up actively and fastest at the external K⁺ concentration used (2.0 mM), partly determines the size of Ca²⁺ influx.     

Effect of Na+, K+, Mg2+ and ATP on the Relative Electrophoretic Mobility of Sugar Beet Membrane Particles with NaK ATPase Activity

Electrophoretic measurements on membrane coated particles were performed with a Zytopherometer. Tris-HCl buffer 0.2 M pH 7.0 at 37°C with addition of different combinations of Na⁺, K⁺, Mg²⁺ and ATP was used as test medium. The membranes were of two types, an untreated preparation with low NaK ATPase activity and a deoxycholate treated preparation with high NaK ATPase activity. There was no marked difference in reaction between the two types of membranes. To both types of membranes Mg²⁺ gave a strong positive and ATP a slight negative addition to the membrane charge. In the presence of ATP Na⁺ gave a higher charge contribution than did K⁺ or a combination of Na⁺ and K⁺. This implies that K⁺ gives a higher affinity for ATP than Na⁺ does and or that ATP mediates a higher affinity for Na⁺ than for K⁺.     

Fluxes of Na+, Rb+ and Cl- Ions in Excised Roots of Sugar Beet Seedlings

Uptake and fluxes of sodium, rubidium (instead of potassium), and chloride ions in segments of 3-week-old sugar beet roots were studied. Radioactive ²²Na, ⁸⁶Rb and ³⁶Cl were used for labelling of the ions. Compartmental analysis was used to obtain the fluxes and concentrations in the cell compartments. The passive or active character of the movements was examined by the Ussing-Teorell equation and compared with electropotential measurements. In the case of sodium, net flux was in the outward direction over both tonoplast and plasmalemma, but the active components were directed away from the cytoplasm. Potassium was close to equilibrium. Chloride was actively transported from the medium to the cytoplasm, and — contrary to observations in other systems — from the vacuole to the cytoplasm. This unusual situation may be caused by a loss of sugar, both by lowering the energy supply and by formation of organic acids.     

Growth and accumulation of N, K+, Ca2+ and Mg2+ in barley exposed to various nutrient regimes and root/shoot temperatures

Six cultivars of spring barley ( Hordeum vulgare L. cvs Salve, Nümberg II, Bomi, Risø 1508, Mona and Sv 73 608) were grown in water culture for three weeks with various combinations of mineral supply and differential roots/shoot temperatures during the growth period. Most important for growth and accumulation of N, K⁺, Ca²⁺ and Mg²⁺ was the mineral supply, followed by the root temperature and the choice of cultivar. Treatments with low mineral supply or low root temperature induced a uniform reduction in growth and accumulation of the ions studied. The effects of low mineral supply and low root temperature on growth and N accumulation was additive, which indicates that these factors exert their influence independently of each other.
Roots grown at 10°C were smaller and Rb⁺(⁸⁶Rb) influx was higher than in roots grown at 20°C. It is suggested that the control of Rb⁺(⁸⁶Rb) influx is affected by the root temperature and the age of the plants. The higher ⁸⁶Rb⁺ (⁸⁶Rb) influx into the low temperature roots could not compensate for the smaller root size. However, the lower total mineral accumulation made up for the needs of the smaller plants and cannot explain the reduction in growth.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

The role of Ca2+ ions in modulating changes induced in bean plants by an excess of Cu2+ ions. Chlorophyll fluorescence measurements

Ca²⁺-dependent influence of excess Cu²⁺ on the photosynthetic akpparatus monitored through chlorophyll fluorescence measurements was investigated in runner bean plants (Phaseolus coccineus L. cv. Pie kny Ja?) at three different growth stages. It was observed that the toxic effect of excess Cu²⁺ on plants depends both on their growth stages and the Ca²⁺ content in the medium. Increased Ca²⁺ content limits Cu²⁺ action on plants at their initial growth stage (I) through: stabilization of the PSII complex (increase of the ratio of variable to minimal fluorescence [F_v/F₀]), improved electron flow and reoxidative processes of the quinone primary electron acceptor of PSII (Q_A) (increase of quantum yield of PSII electron transport [φ_e] and photochemical quenching of fluorescence [q_P] values) and elimination of nonphotochemical energy dissipation (decrease of nonphotochemical fluorescence quenching from the Stern-Volmer equation [NPQ] and fraction of the absorbed light energy not used for photochemistry [LNU] values). At this growth stage excess Cu²⁺ decreases the rates of Q_A reduction as a result of decreased PSII activity at its donor side only at lower Ca²⁺ level. At the intermediate growth stage (II) the plants were less sensitive to Cu²⁺ treatment and also to changed Ca²⁺ content. A weakening of some photochemical processes by excess Cu²⁺ could be observed only at a higher Ca²⁺ dose. At the final growth stage of plants (III) Ca²⁺ ions exerted a decisively different effect on the mechanism of excess Cu²⁺ action on bean plants, visualized by decreased PSII stabilization and utilization of absorbed light energy at increased Ca²⁺ content in the medium.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Effects of Cd2+ and EDTA on young sugar beets (Beta vulgaris). II. Net uptake and distribution of Mg2+, Ca2+ and Fe2+/Fe3+

Levels of Mg²⁺, Ca²⁺ and Fe²⁺/Fe³⁺ were determined in roots and shoots of sugar beet seedlings (Beta vulgaris L. cv. Monohill) cultured for 5 weeks in a complete nutrient solution to which either Cd²⁺ (0, 5 or 50 μM), EDTA (0, 10 or 100 μM) or a combination of both was added. The plants subjected to the various treatments showed a variety of deficiency symptoms. Leaves of the Cd²⁺-treated plants became thin and chlorotic (Mg- and Fe-deficiency symptoms). The plants showed reduced growth and developed only a few brownish roots with short laterals (Ca-deficiency symptoms). EDTA treatment resulted in green, stunted, hard leaves and reduced growth (Ca-deficiency symptoms). The deficiency symptoms observed correspond well with the observed uptake rates and distributions of Mg²⁺, Ca²⁺ and Fe²⁺/Fe³⁺. Increases in either Cd²⁺, EDTA or a combination of both in the growth medium, were correlated with increasing Mg²⁺ levels in the roots and with decreasing Mg²⁺ levels in the shoots. Cd²⁺ alone or in combination with EDTA had little influence on Ca²⁺ levels in the shoots but decreased Ca²⁺ levels in the roots. Thus, Cd²⁺ affects Mg²⁺ and Ca²⁺ transport in opposite ways: Mg²⁺ transport to the shoots is inhibited while that of Ca²⁺ is facilitated. Treatment with EDTA alone did not affect Ca²⁺ concentrations in either the shoots or the roots. Treatment with Cd²⁺ lowered Fe²⁺ concentrations in both roots and shoots.     

Differential effects of Na+, Mg2+, K+, Ca2+ and osmotic stress on the wild type and the NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii

In order to identify physiological components that contribute to salinity tolerance, we compared the effects of Na⁺, Mg²⁺ and K⁺ salts (NaCl, Na₂SO₄, MgCl₂, MgSO₄, KCl and K₂SO₄), Ca₂₊ (CaSO₄), mannitol and melibiose on the wild type and the single-gene NaCl-tolerant mutants stl1 and stl2 of Ceratopteris richardii. Compared with gametophytic growth of the wild type, stl2 showed a low level of tolerance that was restricted to Na⁺ salts and osmotic stress. stl2 exhibited high tolerance to both Na⁺ and Mg²⁺ salts, as well as to osmotic stress. In response to short-term exposure (3 d) to NaCl, accumulation of K⁺ and Na⁺ was similar in the wild type and stl1. In contrast, stl2 accumulated higher levels of K⁺ and lower levels of Na⁺. Ca²⁺ supplementation (1.0 mol m^?3) ameliorated growth inhibition by Na⁺ and Mg²⁺ stress in wild type and stll, but not in stl2. In addition, under Na⁺ stress (175 mol m^?3) wild-type, stll and stl2 gametopbytes maintained higher tissue levels of K⁺ and lower levels of Na⁺ when supplemented with Ca²⁺ (1.0 mol m^?3). stl2 gametophytes were extremely sensitive to K⁺ supplementation. Growth of stl2 was greater than or equal to that of the wild type at trace concentrations of K⁺ but decreased substantially with increasing K⁺ concentration. Supplementation with K⁺ from 0 to 1.85 mol m^?3 alleviated some of the inhibition by 75 mol m^?3 NaCl in the wild type and in stl1. In stl2, growth at 75 mol m^?3 NaCl was similar at 0 and 1.85 mol m^?3 K⁺ supplementation. Although K⁺ supplementation above 1.85 mol m^?3 did not alleviate inhibition of growth by Na⁺ in any genotype, stl2 maintained greater relative tolerance to NaCl at all K⁺ concentrations tested.     

Stimulation of proton extrusion by K+ and divalent cations (Ni2+, Co2+ Zn2+) in maize root segments

Entry of the divalent cations Ni²⁺, Co²⁺ and Zn²⁺ into cells of maize ( Zea mays L. cv. Dekalb XL 85) root tissue is accompanied by an acidification of the incubation medium, a decrease in both the pH of the cell sap and the level of malate in the cells, and by an inhibition of dark fixation of CO₂. K⁺, on the contrary, induces only a very low acidification of the incubation medium, does not change either the pH of the cell sap or the malate level in the cells, and induces an increase in CO₂ dark fixation. Different mechanisms are postulated for the stimulation of proton extrusion by divalent cations and K⁺.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

The effect of Ca2+-stress on fluxes of Ca2+ and K+ in wheat and cucumber

Betula papyrifera Marsh, seedlings adapted very poorly to flooding for up to 60 days. Responses to flooding included increased ethylene production; stomatal closure; leaf senescence; drastic inhibition of shoot growth, cambial growth, and root growth; decay of roots, and death of many seedlings. Flooding inhibited growth of leaves that formed prior to flooding, inhibited formation of new leaves, and induced abscission of old leaves. As a result of extensive leaf abscission, fewer leaves were present after flooding than before flooding was initiated. The drastic reduction in leaf area was associated with greatly decreased growth of the lower stem and roots. No evidence was found of adaptive morphological changes to flooding. The data indicate that intolerance of B. papyrifera seedlings to flooding is an important barrier to regeneration of the species on sites subject to periodic inundation.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Local Anesthetics Inhibit the Ca2+,Mg2+-ATPase Activity of Rat Brain Synaptosomes

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R.I., Epstein P.M., Andrenyak P.M., and Feinstein M.B. (1981) Proc. Natl. Acad. Sci. USA 78, 795-799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.     

Effects of Ca2+ and polyamines on Na+ and Rb+ influx in excised maize roots

When 1 m M spermidine or spermine was included in an absorption solution which contained 20 m M Na⁺ and 1 m M Rb⁺, Na⁺ influx into excised maize roots ( Zea mays L. cv. Golden Cross Bantam) was reduced. Rb⁺ influx was reduced in the presence of spermidine and uneffected in the presence of spermine when compared with control solutions. When 1 m M Ca²⁺ replaced the polyamines, Na⁺ influx was strongly reduced and Rb⁺ influx was promoted. Rb⁺ influx from 1 m M Rb⁺ solutions which did not contain Na⁺ was also promoted by 1 m M Ca²⁺, but was inhibited by 1 m M spermidine. This Ca²⁺ promotion of Rb⁺ influx could be reversed by 10 times greater concentration of spermidine in the absorption solution. H⁺ efflux from excised roots was inhibited by spermidine when compared with Ca²⁺ or control solutions, however, the plasma membrane ATPase was not inhibited by spermidine. It is concluded that external Ca²⁺ plays two separate roles in membrane function, only one of which can be substituted for by polyamines. The first role, maintenance of membrane integrity, can be substituted for by spermidine or spermine. The second function, maintenance of the Rb⁺ transport mechanism, is Ca²⁺ specific and cannot be substituted for by spermidine or spermine. The results of this study are discussed in terms of electrostatic interactions between the plasma membrane and the Ca²⁺ or polyamines.