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⁺.     

Modulation of Na+ transport across leech skin by pesticides and heavy metals

The aim of the present study was to investigate the effects of environmental pollutants, such as heavy metals and pesticides on ion transport across the skin of the leech (Hirudo medicinalis). We wanted to examine the suitability of this epithelium as a model system for studies concerning the mechanisms of toxic action caused by environmental pollutants. For this purpose we performed Ussing chamber experiments to test three representative heavy metals and pesticides, respectively, for their effects on current flow across leech dorsal integument. Two representatives of each substance class showed distinct effects on ion transport across this epithelium. The heavy metal ions Pb²⁺ and Hg²⁺ produced a significant inhibition of amiloride-sensitive Na⁺ transport across leech skin in concentrations below or close to their limiting values in waste water. Therefore, it seems feasible to use leech skin for future investigations of the toxic actions of these heavy metals. The fact that Pb²⁺ and Hg²⁺ exerted their effects only when applied apically points to a specific action of these divalent cations on ion channels in the apical membrane. However, this inhibition does not seem to be a general feature of divalent cations because Cd²⁺ did not influence ion transport across leech skin at all. Since current flow through amiloride-sensitive Na⁺ channels in typical vertebrate tight epithelia is stimulated by numerous divalent cations, the pronounced inhibition of amiloride-sensitive Na⁺ channels in leech skin by Pb²⁺ and Hg²⁺ might lead to a further differentiation of amiloride-sensitive Na⁺ channels. The two widespread pesticides lindane and promecarb exerted their effects only at comparativ high concentrations. This low sensitivity restricts the usefulness of leech skin as a subject for further analysis of toxicity mechanisms, at least for these two pesticides.     

Effects of aluminum chloride on the kinetics of rat cortex synaptosomal ATP diphosphohydrolase 9EC 3.6.1.5)

Aluminum chloride (AlCl₃), a neurotoxic compound, inhibited ATP diphosphohydrolase activity of synaptosomes obtained from cerebral cortex of adult rats. The metal ion significantly inhibited ATPase and ADPase activities of the enzyme at all concentrations tested in vitro (0.01, 0.05, 0.5, 5 and 10 mM) in the presence of 1.5 mM calcium. When tested in the absence of Ca²⁺, and with increasing amounts of Al³⁺, enzyme activity remained below basal levels, suggesting that the trivalent cation Al³⁺ is not a substitute for the divalent cation Ca²⁺ in ATP-Ca²⁺ and ADP-Ca²⁺ complexes. The Al³⁺ inhibition was competitive with respect to Ca²⁺. The enzyme inhibition was reversed by the addition of deferoxamine (DFO). NaF significantly inhibited ATP diphosphohydrolase activity, and this inhibition was reversed by the addition of Ca²⁺ to the medium. Such inhibition was not potentiated by AlF₄, which is an inhibitor of cation-transport ATPases.     

Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three‐metal ion assisted catalysis and lithium inhibition

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg²⁺ for activity and binds three to four ions tightly in the absence of ligands: K_D = 0.8 μM for one ion with a K_D of 38 μM for the other Mg²⁺ ions. However, the enzyme requires 5–10 mM Mg²⁺ for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic K_D for Mg²⁺; D26A, E39A, and E41A showed no significant change in the Mg²⁺ requirement for optimal activity. D38A also showed an increased K_m, but little effect on k_cat. This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg²⁺ in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC₅₀～250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li⁺ with an IC₅₀ of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily.     

Combined actions of Pb2+, Zn2+, and Al3+ on voltage-activated calcium channel currents

Summary 1. Whole-cell patch clamp experiments were performed on rat dorsal root ganglion (DRG) neurons to investigate the actions of various combinations of Pb²⁺, Zn²⁺, and Al³⁺ on voltage-activated calcium channel currents (VACCCs).2. Each of these metals has been shown to reduce VACCCs.3. We investigated the effects of simultaneous application of two cations in the range of their IC₅₀ values. For all possible combinations (Pb²⁺/Zu²⁺, Zn²⁺/Al³⁺, Al³⁺/Pb²⁺), independent of the order of application, we found additive actions on VACCCs.4. We observed a 75% (±9%) block of the control current when two cations were applied simultaneously. This observation is consistent with both, an action of two metals at the same site as well as independent actions at different locations of the ion channel.5. The additivity of the effects should be taken into account for questions of public health and the assessment of threshold limits in cases of environmental contamination.     

重金属离子对猪红细胞膜Ca~(2+)-ATP酶的作用

猪红细胞膜Ca~(2+)-ATP酶是一种钙调蛋白(CaM)依赖酶,其活力又依赖巯基的完整性。实验应用Ca~(2+)-ATP酶这一模型体系观察到重金属离子,Pb~(2+)、Cd~(2+)和Hg~(2+)都能替代Ca~(2+),激活CaM,从而激活Ca~(2+)-ATP酶;其最大刺激活力分别为85％、80％和30％,半刺激浓度分别为32、27和0.7μmol/L。当三种重金属离子的浓度增加时,则与Ca~(2+)-ATP酶的巯基结合,抑制酶的活力,Pb2~(2+)、Cd~(2+)和Hg~(2+)的半抑制浓度分别为370、440和2μmol/L。抑制作用为渐进性过程,而刺激作用为即时效应。抑制作用可为巯基化物,特别是二巯基化物所逆转。研究结果提示,CaM可能是重金属中毒最初作用的靶分子,而重金属中毒不仅使CaM“开关”失灵,还可能导致细胞内Ca~(2+)的调节全面失控。     

Structure determination and refinement of the Al complex of the D254,256E mutant of Arthrobacter -xylose isomerase at 2.40 Å resolution. Further evidence for inhibitor-induced metal ion movement

The structure of the D254,256E double mutant of Arthrobacter xylose isomerase with Al³⁺ at both metal-binding sites was determined by the molecular replacement method at a conventional R-factor of 0.179. Binding of the two Al³⁺ does not alter the overall structure significantly. However, there are local rearrangements in the octahedral co-ordination sphere of the Al³⁺. The inhibitor molecule moves somewhat away from the active site. Furthermore, evidence was revealed for metal ion movement from site 2₁ to site 2₂ upon double mutation. Xylose isomerase requires two divalent metal cations for activation. The catalytic metal ion is translocated 1.8 Å away from its initial position during the catalytic reaction. The fact that both activating and inactivating metals (including Al³⁺) were found exclusively at a single location in the double mutant was an indication that the consequently missing shuttle may account for the crippled catalytic efficiency.     

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.     

Molecular dynamics simulation study for ion mobility of alkali earth metal cations in water at 25°C

We present the results of molecular dynamics simulations for alkali earth metal cations (Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺) in an aqueous solution at 25°C using the extended simple point charge water potential with Ewald summation. The ion mobilities (defined by u_i = D_iz_iF/RT) obtained from the simulations are in good accord with the experimental measures. The strong divalent cation–water interactions explain well the static and dynamic properties of the alkali earth metal cations. The classical solvent-berg picture describes the ion mobilities of these cations in water adequately.     

Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor

Herein, we identify the coordination environment of Cu²⁺ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²⁺ and Cu²⁺ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn²⁺ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu²⁺ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu²⁺ coordination and stoichiometry. We show that Cu²⁺ has one binding site per α1 subunit, and that five Cu²⁺ can be coordinated per GlyR. Cu²⁺ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu²⁺ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn²⁺ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.     

Toxicity of chromium and tin toAnabaena doliolum Interaction with bivalent cations

Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity ofAnabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca²⁺, Mg²⁺ and Mn²⁺, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca²⁺ > Mg²⁺ > Mn²⁺. However, the sequence of hierarchy was Mg²⁺ > Ca²⁺ > Mn²⁺ for carbon fixation and Mn²⁺ > Mg²⁺ > Ca²⁺ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth,¹⁴CO₂ uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity ofA. doliolum when Ni²⁺, Co²⁺ and Zn²⁺ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni²⁺ > Co²⁺ > Zn²⁺. Among all the interacting cations, Ca²⁺, Mg²⁺ and Mn²⁺ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni²⁺, CO²⁺ and Zn²⁺ showed synergistic inhibition which potentiating the toxicity of test metals in the N₂-fixing cyanobacteriumA. doliolum. It is evident from the present study that bivalent cations, viz. Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Zn²⁺, may appreciably regulate the toxicity of heavy metals in N₂-fixing cyanobacteria if present in aquatic media.     

Spatiotemporal Alterations of the NO/NOS Neuronal Pools Following Transient Abdominal Aorta Occlusion: Morphological and Biochemical Studies in the Rabbit

1. Brief interruption of spinal cord blood flow resulting from transient abdominal aortic occlusion may lead to degeneration of specific spinal cord neurons and to irreversible loss of neurological function. The alteration of nitric oxide/nitric oxide synthase (NO/NOS) pool occurring after ischemic insult may play a protective or destructive role in neuronal survival of affected spinal cord segments.2. In the present study, the spatiotemporal changes of NOS following transient ischemia were evaluated by investigating neuronal NOS immunoreactivity (nNOS-IR), reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, and calcium-dependent NOS (cNOS) conversion of [³H] l-arginine to [³H] l-citrulline.3. The greatest levels of these enzymes and activities were detected in the dorsal horn, which appeared to be most resistant to ischemia. In that area, the first significant increase in NADPHd staining and cNOS catalytic activity was found immediately after a 15-min ischemic insult.4. Increases in the ventral horn were observed later (i.e., after a 24-h reperfusion period). While the most intense increase in nNOS-IR was detected in surviving motoneurons of animals with a shorter ischemic insult (13 min), the greatest increase of cNOS catalytic activity and NADPHd staining of the endothelial cells was found after stronger insult (15 min).5. Given that the highest levels of nNOS, NADPHd, and cNOS were found in the ischemia-resistant dorsal horn, and nNOS-IR in surviving motoneurons, it is possible that NO production may play a neuroprotective role in ischemic/reperfusion injury.     

Binding of metal ions toE. coli RNase HI observed by1H−15N heteronuclear 2D NMR

Summary The divalent metal ion binding site and binding constant of ribonuclease HI fromEscherichia coli were investigated by observing chemical shift changes using¹H–¹⁵N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by¹⁵N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide¹⁵N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg²⁺, Ca²⁺, or Ba²⁺. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (K_D=2–4 mM for the current divalent ions).     

Effects of cadmium on the cuttlefish Sepia pharaonis’ arginine kinase: unfolding kinetics integrated with computational simulations

Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd²⁺ on the cuttlefish Sepia pharaonis’ arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd²⁺ effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd²⁺ induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd²⁺ induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd²⁺ showed that Cd²⁺ partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd²⁺-induced inactivation of SPAK. These results demonstrate the effect of Cd²⁺ on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd²⁺ in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.     

La3+ stimulate the activity of calcineurin in two different ways

It is well known that the activity of calcineurin (CaN) could be modulated by several transitional metal ions. In the present work, the effects of a calcium analog, lanthanum ion (La³⁺), on the activity of CaN were studied. It was found that La³⁺ exerted multiple effects on CaN activity. La³⁺ could stimulate CaN in the absence of calmodulin (CaM); whereas at low concentrations of La³⁺, there was a slight inhibition of activation of CaN in the presence of CaM. Competitive experiments and limited trypsin proteolysis confirmed that La³⁺ did not act on the catalytic core of CaN, but exerted its effect through direct action on the CaN regulatory domain similar to Mg²⁺. In activity titration and spot blotting studies, La³⁺-containing CaM complexes were less effective in stimulating CaN than Ca²⁺ or Mn²⁺-containing CaM; however, the binding affinity of these metal–CaM complexes to CaN was similar. These effects of La³⁺ on CaN activity are unique among metal ions and may provide clues to understand the biological effects of La³⁺.     

酿酒酵母细胞中内质网应激与未折叠蛋白反应的研究进展

内质网应激激活的未折叠蛋白反应(Unfolded protein response,UPR)途径在酿酒酵母和哺乳动物细胞中是非常保守的。内质网(Endoplasmic reticulum,ER)是蛋白质合成、折叠和修饰的细胞器,也是贮存钙的主要场所之一。酵母细胞内质网钙平衡与UPR的作用是相互的;两个MAPK途径——HOG途径和CWI途径都是细胞应答内质网应激压力时生存所必需的;重金属镉离子能够激活UPR途径,它通过激活钙离子通道Cch1/Mid1进入细胞影响钙离子的功能。本文结合最新研究进展对酿酒酵母细胞中的两个MAPK途径、镉离子和钙离子稳态与内质网应激激活的UPR途径之间相互关系进行综述。     

Use of the coulombic interactions of the lanthanide series to identify two classes of Ca2+ binding sites in mitochondria

The degree of inhibition of respiration-dependent vs respiration-independent Ca²⁺ binding by rat liver mitochondria by different members of the lanthanide family was used to establish the existence of two different classes of Ca²⁺ binding sites. The distinction is based on the differences in cation:site interactions between the two classes of sites and the members of the lanthanide series. Lanthanide inhibition of respiration-dependent Ca²⁺ uptake suggests that the binding site is specific for the calcium ion. Those members of the lanthanide family whose ionic radii are nearer that of Ca²⁺ are the best inhibitors. The inhibition of respiration-independent Ca²⁺ binding is much different, indicating non-specific cation absorption.     

Ca2+ effects on Fe(II) interactions with Mn-binding sites in Mn-depleted oxygen-evolving complexes of photosystem II and on Fe replacement of Mn in Mn-containing,Ca-depleted complexes

Fe(II) cations bind with high efficiency and specificity at the high-affinity (HA), Mn-binding site (termed the “blocking effect” since Fe blocks further electron donation to the site) of the oxygen-evolving complex (OEC) in Mn-depleted, photosystem II (PSII) membrane fragments (Semin et al. in Biochemistry 41:5854, 2002). Furthermore, Fe(II) cations can substitute for 1 or 2Mn cations (pH dependent) in Ca-depleted PSII membranes (Semin et al. in Journal of Bioenergetics and Biomembranes 48:227, 2016; Semin et al. in Journal of Photochemistry and Photobiology B 178:192, 2018). In the current study, we examined the effect of Ca²⁺ cations on the interaction of Fe(II) ions with Mn-depleted [PSII(-Mn)] and Ca-depleted [PSII(-Ca)] photosystem II membranes. We found that Ca²⁺ cations (about 50 mM) inhibit the light-dependent oxidation of Fe(II) (5 µM) by about 25% in PSII(-Mn) membranes, whereas inhibition of the blocking process is greater at about 40%. Blocking of the HA site by Fe cations also decreases the rate of charge recombination between Q_A^? and Y_Z^?+ from t_1/2?=?30 ms to 46 ms. However, Ca²⁺ does not affect the rate during the blocking process. An Fe(II) cation (20 µM) replaces 1Mn cation in the Mn₄CaO₅ catalytic cluster of PSII(-Ca) membranes at pH 5.7 but 2 Mn cations at pH 6.5. In the presence of Ca²⁺ (10 mM) during the substitution process, Fe(II) is not able to extract Mn at pH 5.7 and extracts only 1Mn at pH 6.5 (instead of two without Ca²⁺). Measurements of fluorescence induction kinetics support these observations. Inhibition of Mn substitution with Fe(II) cations in the OEC only occurs with Ca²⁺ and Sr²⁺ cations, which are also able to restore oxygen evolution in PSII(-Ca) samples. Nonactive cations like La³⁺, Ni²⁺, Cd²⁺, and Mg²⁺ have no influence on the replacement of Mn with Fe. These results show that the location and/or ligand composition of one Mn cation in the Mn₄CaO₅ cluster is strongly affected by calcium depletion or rebinding and that bound calcium affects the redox potential of the extractable Mn4 cation in the OEC, making it resistant to reduction.

     

Quantification of single-stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis: part I

The interactions between oligonucleotides and inorganic cations have been measured by capillary zone electrophoresis. With increasing concentrations of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺ and Ni²⁺) in the running buffer, the migration behavior was evaluated by calculation of the binding constants. Besides these fundamental studies of binding equilibria, different buffer components, tris(hydroxymethyl)aminomethane and 3-(N-morpholino)propanesulfonic acid, have been investigated and their effects on metal ion binding quantified.