Stability of Ca2+-, Cd2+-, Cu2+-[illite-humic] complexes and pH influence

Decomposition of fresh plant residues in soil is expected to produce humic fractions varying in molecular size. It was hypothesized that metal adsorption by soil, to some degree, will depend on humic acid content and molecular size. The latter is expected to vary in number and type of functional groups. In this study, illite-humic complexes were used to evaluate Ca²⁺, Cd²⁺, and Cu²⁺ adsorption and how this adsorption was affected by humic acids, differing in molecular size, under various pH values. Potentiometric titration using ion-selective electrodes with a stop-and-go procedure was employed to evaluate metal-[illite-humic] complex formation. The results showed that illite-humic complexes exhibited at least two types of metal-ion adsorption sites (low and high affinity) and molecular size of humic fractions had a large potential influence on total metal adsorption but a relatively smaller influence on metal-complex stability. Relative strength of metal-ion-[illite-humic] complexes followed the order of Cu²⁺>Cd²⁺>Ca²⁺ and were affected by pH, especially for low metal-ion affinity sites. Magnitude of metal-[illite-humic] stability constants, depending on molecular size of humic fraction and pH, varied on a log-scale from 3.52 to 4.21 for Ca²⁺, 4.38 to 5.18 for Cd²⁺and from 5.23 to 5.83 for Cu²⁺. There was an approximate 5-fold difference in these stability constants between the three different sizes of humic fractions. The larger the humic fraction, the lower the metal-[illite-humic] stability constant. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date. This revised version was published online in June 2006 with corrections to the Cover Date.     

Metal-humic complexes and plant micronutrient uptake: a study based on different plant species cultivated in diverse soil types

There are several studies in the literature dealing with the effect of metal-humic complexes on plant metal uptake, but none of them correlate the physicochemical properties of the complexes with agronomic results. Our study covers both aspects under various experimental conditions. A humic extract (SHE) obtained from a sapric peat was selected for preparing the metal–humic complexes used in plant experiments. Fe–, Zn– and Cu–humic complexes with a reaction stoichiometry of 2:0.25 (humic:metal, w/w) were chosen after studying their stability and solubility with respect to pH (6–9) and the humic:metal reaction stoichiometry. Wheat and alfalfa plants were greenhouse cultured in pots containing one of three model soils: an acid, sandy soil and two alkaline, calcareous soils. Treatments were: control (no additions), SHE (53 mg kg^–1 of SHE), and metal (Cu, Zn and Fe)–SHE complexes (2.5 and 5 mg kg^–1 of metal rate and a SHE concentration to make 53 mg kg ^–1). Cu- and Zn–humic complexes significantly (p0.05) increased the plant uptake and the DTPA-extractable soil fraction of complexed micronutrients in most plant–soil systems. However, these effects were associated with significant increases (p0.05) of shoot and root dry weight only in alfalfa plants. In wheat, significant increases of root and shoot dry matter were only observed in the Cu–humic treated plants growing in the acid soil, where Cu deficiency was more intense. The Fe–humic complex did not increase Fe plant assimilation in any plant–soil system, but SHE increased Fe-uptake and/or DTPA-extractable soil Fe in the wheat–calcareous soil systems. These results, taken together with those obtained from the study of the pH- and SHE:metal ratio-dependent SHE complex solubility and stability, highlight the importance of the humic:Fe complex stoichiometry on iron bioavailability as a result of its influence on complex solubility.     

Actions of cadmium on basolateral plasma membrane proteins involved in calcium uptake by fish intestine

Summary The inhibition of Ca^2–-ATPase, (Na⁺+K⁺)-ATPase and Na⁺/Ca²⁺ exchange by Cd²⁺ was studied in fish intestinal basolateral plasma membrane preparations. ATP driven ⁴⁵Ca²⁺ uptake into inside-out membrane vesicles displayed a K _m for Ca²⁺ of 88±17 nm, and was extremely sensitive to Cd²⁺ with an IC₅₀ of 8.2±3.0 pM Cd²⁺, indicating an inhibition via the Ca²⁺ site. (Na⁺+K⁺)-ATPase activity was half-maximally inhibited by micromolar amounts of Cd²⁺, displaying an IC₅₀ of 2.6±0.6 m Cd²⁺. Cd²⁺ ions apparently compete for the Mg²⁺ site of the (Na^– +K⁺)-ATPase. The Na⁺/Ca²⁺ exchanger was inhibited by Cd²⁺ with an IC₅₀ of 73±11 nm. Cd²⁺ is a competitive inhibitor of the exchanger via an interaction with the Ca²⁺ site (K _i = 11 nm). Bepridil, a Na⁺ site specific inhibitor of Na⁺/Ca²⁺ exchange, induced an additional inhibition, but did not change the K _i of Cd²⁺. Also, Cd²⁺ is exchanged against Ca²⁺, albeit to a lesser extent than Ca²⁺. The exchanger is only partly blocked by the binding of Cd²⁺. In vivo cadmium that has entered the enterocyte may be shuttled across the basolateral plasma membrane by the Na⁺/Ca²⁺ exchanger. We conclude that intracellular Cd²⁺ ions will inhibit plasma membrane proteins predominantly via a specific interaction with divalent metal ion sites.We would like to thank Dr. D. Fackre (University of Alberta, Canada) for stimulating discussions and Mr. F.A.T. Spanings (University of Nijmegen, The Netherlands) for excellent fish husbandry. The fura-2 measurements of intracellular Ca²⁺ concentrations in tilapia enterocytes were carried out in the Department of Physiology, School of Medicine, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Th.J.M. Schoenmakers and G. Flik were supported by travel grants from the Foundation for Fundamental Biological Research (BION) and the Netherlands Organization for Scientific Research (NWO).     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Zinc inhibition of chloride efflux from skeletal muscle ofRana pipiens and its modification by external pH and chloride activity

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases. In depolarized muscles fromRana pipiens, the pH-dependent Cl^– efflux has an apparent pK _a near 6.4.The reduction of Cl^– efflux by external Zn²⁺ was determined at different external pHs and chloride activities. The effect of external chloride activity on the pH-dependent Cl^– efflux was also examined.At pH 6.5 and a membrane potential of –22 mV, increasing external Cl^– activity from 0.108 to 0.28m decreased inhibition of the pH-dependent Cl^– efflux at all activities of Zn²⁺. The Zn²⁺ activity needed to reduce Cl^– efflux by half increased from 0.39×10^–3 to 2.09×10^–3 m. By contrast, external Cl^– activity had no measurable effect on the apparent pK _a of the pH-dependent efflux.At constant Cl^– activity less than 0.21m, increasing external pH from 6.5 to 7.5 decreased inhibition by low Zn²⁺ activities with either a slight increase or no change in the Zn²⁺ activity producing half-inhibition. In other words, for relatively low Cl^– activities, protection against inhibition of Cl^– efflux by low Zn²⁺ activities was obtained by raising, not lowering, external pH; this is not what is expected if H⁺ and Zn²⁺ ions compete at the same site to produce inhibition of Cl^– efflux. We conclude that Zn²⁺ and low pH inhibit Cl^– efflux by separate and distinct mechanisms.By contrast, the protection against Zn²⁺ inhibition produced by high external Cl^– activity (0.28m) was partially reversed by raising external pH from 6.5 to 7.5 at all Zn²⁺ activities. The half-inhibition Zn²⁺ activity decreased from 2.09×10^–3 to 0.68×10^–3 m.The results can be simulated quantitatively by a model in which single Cl^– channel elements are in equilibrium with sextets of associated single-channel elements, each sextet having a conductance six times that of a single-channel element. The association into sextets is promoted by OH^– or Cl^– binding to a control site on the single-channel elements. Both the single Cl^– channel element and the sextet of Cl^– channel elements are closed when this same control site instead binds ZnOH⁺. The sextet has a much higher affinity for ZnOH⁺ than does the single Cl^– channel element.     

UPTAKE OF CADMIUM BY FRESHWATER GREEN ALGAE: EFFECTS OF PH AND AQUATIC HUMIC SUBSTANCES1

The effects of humic substances and low pH on short‐term Cd uptake by Pseudokirchneriella subcapitata (Korshikov) Hindak and Chlamydomonas reinhardtii Dang were investigated under defined exposure conditions. The uptake experiments were run in the presence of either a synthetic organic ligand (nitrilotriacetate) or natural organic ligands (Suwannee River fulvic or humic acid). An ion‐exchange method was used to measure the free Cd²⁺ concentrations in the exposure solutions. At pH 5, measured free Cd²⁺ concentrations agreed with estimations made using the geochemical equilibrium model WHAM, but at pH 7 the model overestimated complexation by both Suwannee River fulvic and humic acids compared with the ion‐exchange measurements. Consistent with the metal internalization step being rate limiting for overall short‐term uptake, intracellular Cd uptake was linear for exposure times less than 20 min at pH 5 or pH 7 for both algal species. After taking into account complexation of Cd in solution, Suwannee River humic substances had no additional effects on cadmium uptake at pH 7, as would be predicted by the free ion model. This absence of effects other than complexation persisted at pH 5, where the tendency of humic substances to adsorb to the algal cell surface is favored. Changes in pH strongly influenced Cd uptake, with the intracellular flux of Cd being at least 20 times lower at pH 5 than at pH 7 for P. subcapitata. Our results support models such as the free ion model or the biotic ligand model, in which humic substances act indirectly on Cd uptake by reducing the bioavailability of Cd by complexation in solution.     

Metal-ion-dependent hydrophobic-interaction chromatography of α-lactalbumins

α-Lactalbumins from bovine, human, goat, sheep, and horse milk bind to phenyl-Sepharose in the presence of EDTA and can be eluted by addition of Ca²⁺ (0.001–100 m ). This property has been utilized to purify these proteins in a one-step purification from milk whey. α-Lactalbumin purified in this manner has the same ultraviolet and proton nuclear magnetic resonance spectra as that purified by other methods. Using binding to phenyl-Sepharose as an assay, the conformation of bovine α-lactalbumin upon the addition of several metal ions that are known to interact with this protein was investigated. Lanthanides, Mn²⁺, Mg²⁺, and Cd²⁺ can substitute for Ca²⁺, whereas Zn²⁺, Al³⁺, and Co²⁺ cannot. Surprisingly, whereas lower concentrations of La³⁺, Mn²⁺, and Cd²⁺ (1 m and less) caused elution from the hydrophobic support, higher concentrations (10 m ) were ineffective. These observations can be rationalized assuming the presence of two distinct metal-ion binding sites with different specificities.     

Ca2+-activated Cl− channel in plasmalemma ofNitellopsis obtusa

Summary The mechanisms of Cl^–-channel activation in the plasmalemma ofNitellopsis obtusa was studied by measuring both the transient inward current under voltage clamp and Cl^– efflux during the action potential. 9-anthracenecarboxylic acid (A-9-C) at 1.0mm inhibited both the transient inward current and the Cl^– efflux, but did not uncouple the sudden cessation of the cytoplasmic streaming. Since this excitation-cessation coupling is caused by a transient increase in the cytoplasmic Ca²⁺ concentration, these results suggest that A-9-C inhibited not the Ca²⁺ channel but specifically the Cl^– channel. The following results were found between the Ca²⁺-channel activation and the Cl^–-channel activation: (1) The Ca²⁺-channel blocker La³⁺ uncoupled the excitation-cessation coupling and inhibited both the transient inward current and the Cl^– efflux, although the Cl^–-channel blocker A-9-C did not affect the excitation-cessation coupling. (2) The Cl^– efflux was greatly reduced by depletion of Ca²⁺ from the external solution and restored by an increase in the external Ca²⁺ concentration. (3) An increase in the external ionic, strength which increases Ca²⁺ entry (T. Shiina & M. Tazawa,J. Membrane Biol. 96:263–276, 1987) enhanced the Cl^– efflux. (4) Mg²⁺, which cannot pass through the Ca²⁺ channel, reduced both the transient inward current and the Cl^– efflux. (5) Although Sr²⁺ can pass through the plasmalemma Ca²⁺ channel, Cl^–-channel activation by Sr²⁺ was only partial. These findings support the hypothesis that voltage-dependent Ca²⁺-channel activation, which increases the free Ca²⁺ concentration in the cytoplasm, is necessary for the subsequent Cl^–-channel activation.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

Activation by hyperpolarization and atypical osmosensitivity of a Cl− current in rat osteoblastic cells

During whole-cell recording of rat osteoblastic cells with high-Cl^– internal solutions, 10 sec hyperpolarizing jumps from 0 mV induce a slow inward current relaxation, which is shown to be carried by hyperpolarization-activated Cl^– channels. This relaxation increases and becomes faster with stronger hyperpolarizations. It is insensitive to Cs⁺ ions but is blocked in a voltage-dependent manner by 4,4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS) 1 mm and is reduced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) 0.1 mm. Cd²⁺ ions are potent blockers of this current, blocking completely above 300 m. The amplitude of the Cl^– current activated by a given hyperpolarization increases during the first 10–20 min of whole-cell recording. This evolution and the fact that some recently cloned Cl^– channels have been reported to be activated both by hyperpolarization and by external hyposmolarity led us to investigate the effects of external osmolarity. Reducing the external osmolarity induces a large Cl^– current. However, this hyposmolarity-induced Cl^– current and the hyperpolarization-activated Cl^– current are shown to be distinct; 1,9-dideoxy forskolin selectively blocks the hyposmolarity-activated current. We show that the hyperpolarization-activated Cl^– current is osmosensitive, but in an unusual way: it is reduced by external hyposmolarity and is increased by external hyperosmolarity. Furthermore, these modulations are more pronounced for small hyperpolarizations. The osmosensitivity of the hyperpolarization-activated Cl^– current suggests a mechanosensitivity (activation by positive external pressure) that is likely to be physiologically important to bone cells.We wish to thank P. Ascher and B. Barbour for useful comments.     

Hg2+-induced leakage of electrolytes and inhibition of NO3 − utilization inNostoc calcicola

Summary The effect of mercury (Hg²⁺) in the absence and presence of methylmercury (CH₃Hg⁺), cadmium (Cd²⁺), copper (Cu²⁺), nickel (Ni²⁺) and calcium (Ca²⁺) on Nostoc calcicola Bréb. has been studied in terms of electrolyte leakage, NO₃ ^– uptake and in vivo nitrate reductase (NR) activity to discover any possible correlation among such parameters under Hg²⁺ stress. Leakage of electrolytes from Hg²⁺-treated cyanobacterial cells was directly proportional to Hg²⁺ concentrations and exposure time. In comparison to NO₃ ^– uptake, an about 60-fold slower rate of NR activity was observed in the untreated cultures, the former being five times more Hg²⁺-sensitive. A non-competitive synergistic interaction of Hg²⁺ with CH₃Hg⁺ or Cd²⁺ and antagonistic with that of Ni²⁺ or Ca²⁺ has been observed for both the processes of NO₃ ^– utilization. The antagonistic interaction of Cu²⁺ with Hg²⁺ in terms of NO₃ ^– uptake and synergistic with respect to NR activity, has been attributed to the dual bonding preference of Cu²⁺ for cellular ligands. These findings suggest that (a) a statistically significant correlation exists among such parameters; (b) Hg²⁺ predominantly attacks the cyanobacterial cell membrane; (c) Hg²⁺ inhibits NO₃ ^– utilization; (d) the presence of other cations increases or decreases the inhibitory actions of Hg²⁺.     

Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.     

Identification of the cardiac sarcolemmal Na+−Ca2+ exchanger using monoclonal antibodies

Summary We have previously partially purified the sarcolemmal Na⁺–Ca²⁺ exchange protein and produced rabbit polyclonal antibodies to the exchanger (Philipson, K. D., Longoni, S., Ward, R. 1988.Biochim. Biophys.Acta 945:298–306). We now describe the generation of three stable murine hybridoma lines which secrete monoclonal antibodies (MAb's) to the exchanger. These MAb's immunoprecipitate 50–75% of solubilized Na⁺–Ca²⁺ exchange activity. The MAb's appear to be reactive with native conformation-dependent expitopes on the Na⁺–Ca²⁺ exchanger since they do not react on immunoblots. An indirect method was used to identify Na⁺–Ca²⁺ exchange proteins. A column containing Na⁺–Ca²⁺ exchanger immobilized by MAb's was used to affinity purify the rabbit polyclonal antibody. The affinity-purified polyclonal antibody reacted with proteinsof, apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our prevous association of Na⁺–Ca²⁺ exchange with these proteins.     

Effect of Cd and Hg on the Activity of Na/K-ATPase and Mg-ATPase Adsorbed on Polystyrene Microtiter Plates

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4°C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na⁺/K⁺-ATPase and Mg²⁺-ATPase, retained 70–80% activity after the adsorption. In addition, adsorption stabilizes Na⁺/K⁺-ATPase and Mg²⁺-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na⁺/K⁺-ATPase and Mg²⁺-ATPase and their sensitivity to and mechanism of Cd²⁺- or Hg²⁺-induced inhibition. The only exception is the “high affinity” Mg²⁺-ATPase moiety, whose affinity for ATP and sensitivity toward Cd²⁺ were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Detection of the Gua/Cyt-to-Cyt/Gua mutation in a Gua/Cyt-lined sequence using Zn2+-cyclen polyacrylamide gel electrophoresis

We previously reported a method for the detection of single-nucleotide polymorphisms by polyacrylamide gel electrophoresis (PAGE) with an additive Zn²⁺–cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane), called Zn²⁺–cyclen–PAGE. The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE that is due to Zn²⁺–cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. In combination with a heteroduplexing technique, the method is more accurate, as shown by clear gel-shifting bands. However, the question remains as to whether the Gua/Cyt-to-Cyt/Gua mutation, which is far apart from the Thy/Ade base (i.e., in a Gua/Cyt-lined sequence), can be detected by this Thy-dependent method. In this study, we determined the potency of Zn²⁺–cyclen–PAGE for the detection of the Gua/Cyt-to-Cyt/Gua single substitutions in some artificial Gua/Cyt-lined sequences derived from a human cardiac sodium channel gene, SCN5A. All Gua/Cyt-to-Cyt/Gua substitutions in the 28-set samples tested, which are 1 to 10 bases away from the nearest Thy/Ade, were successfully detected by designing DNA fragments of the appropriate length.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Composition and metal ion complexation behavour of humic fractions derived from corn tissue

Decomposition of fresh plant residues produces humic fractions with different molecular size and composition. It was hypothesized that the functional group-type and content of humic fractions depended on molecular size, which was expected to influence heavy-metal complexation behavior. In this study, corn (Zea maysL.) stalks and leaves were collected from the field and decomposed for an 8-month period to produce humic substances which were separated into three water soluble fractions, HF1, HF2 and HF3, from highest to lowest relative molecular size. Functional group determination showed that total, carboxylic and phenolic OH acidity increased as relative molecular size of humic fractions decreased. Furthermore, C/O ratios decreased, whereas N/C and H/C ratios remained relatively unaffected as relative molecular size of humic fractions decreased. Formation of Ca²⁺, Cd²⁺ and Cu²⁺ -humic fraction complexes and how these complexes were affected by pH and relative (humic fraction) molecular size were studied using potentiometric titration. Metal-humic complexes exhibited at least two types of sites with respect to Ca²⁺, Cd²⁺ and Cu²⁺ complexation. Relative molecular size had a large significant influence on total metal-ion complexation, but it had a relatively small influence on complex stability at low levels of metal-ion complexation. Strength of metal-ion humic complexes followed the order Cu²⁺ > Cd²⁺ > Ca²⁺ and was affected by pH, especially for low affinity sites. Carboxylic and phenolic OH groups were most likely involved in complex formation. Magnitude of the metal-humic formation constants at the lowest equilibrium metal-ion concentration, under the various pH values tested, varied from 5.39 to 5.90 for Ca²⁺, from 5.36 to 6.01 for Cd²⁺ and from 6.93 to 7.71 for Cu²⁺. Furthermore, the formation constants appeared to be positively influenced by decreasing molecular size of water-soluble humic fraction, and increasing pH. These results inferred that soil management practices causing build-up of humic substances would affect mobility and bioavailability of metal-ions.     

Differences in the binding modes of phytochelatin to cadmium (II) and Zinc(II) ions

An ¹H NMR (nuclear magnetic resonance) spectroscopic structural analysis of Cd²⁺ complexes formed with the pentapeptide phytochelatin, (NH₃)⁺−(ψ-Glu-Cys)₂−Gly−COO−(PC₂), at a pH of 7.5 showed that the two thiol groups of the Cys residues and either the carbonyl or amide group of the peptide bond between Glu1 and Cys1 act as possible donor groups in the complexes at Cd²⁺/PC₂ ratios up to 0.4. As the ratio increases, the carboxylate group of Glu2 and either the carbonyl or amide group of the peptide bond between Cys1 and Glu2 comes to serve as a donor group. The manner in which Cd²⁺ forms complexes with PC₂ is distinctly different from Zn²⁺ and might account for the role of phytochelatin in Cd²⁺ detoxification. Electron absorption spectrometry demonstrated that the Cd²⁺ complexes are coordinated in a tetrahedral fashion by four thiol groups and that several sulfur atoms might bridge Cd²⁺ ions, resulting in the formation of polynuclear complexes. This contrasts with Zn²⁺ complex formation, which consists exclusively of a 1:1 complex.     

Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

Summary Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na⁺–Ca²⁺ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na⁺–Ca²⁺ exchange. The treatment with dithiothreitol resulted in a decrease inV _max values but did not alter theK _m for Ca²⁺ for the Na²⁺–Ca²⁺ exchange reaction. If Na⁺ replaced K⁺ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na⁺–Ca²⁺ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N-ethylmaleimide, were capable of altering Na⁺–Ca²⁺ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na⁺–Ca²⁺ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na⁺–Ca²⁺ exchange, depending on whether Na⁺ or K⁺ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfurcontaining residues interact with a Na⁺-binding site for Na⁺–Ca²⁺ exchange in cardiac sarcolemmal vesicles.