Expression of novel cytosolic malate dehydrogenases (cMDH) in Lupinus angustifolius nodules during phosphorus starvation

During P deficiency, the increased activity of malate dehydrogenase (MDH, EC 1.1.1.37) can lead to malate accumulation. Cytosolic- and nodule-enhanced MDH (cMDH and neMDH, respectively) are known isoforms, which contribute to MDH activity in root nodules. The aim of this study was to investigate the role of the cMDH isoforms in nodule malate supply under P deficiency. Nodulated lupins (Lupinus angustifolius var. Tanjil) were hydroponically grown at adequate P (+P) or low P (−P). Total P concentration in nodules decreased under P deficiency, which coincided with an increase in total MDH activity. A consequence of higher MDH activity was the enhanced accumulation of malate derived from dark CO₂ fixation via PEPC and not from pyruvate. Although no measurable neMDH presence could be detected via PCR, gene-specific primers detected two 1 kb amplicons of cMDH, designated LangMDH1 (corresponding to +P, HQ690186) and LangMDH2 (corresponding to −P, HQ690187), respectively. Sequencing analyses of these cMDH amplicons showed them to be 96% identical on an amino acid level. There was a high degree of diversification between proteins detected in this study and other known MDH proteins, particularly those from other leguminous plants. Enhanced malate synthesis in P-deficient nodules was achieved via increased anaplerotic CO₂ fixation and subsequent higher MDH activities. Novel isoforms of cytosolic MDH may be involved, as shown by gene expression of specific genes under P deficiency.     

Simultaneous purification of L-malate dehydrogenase and L-lactate dehydrogenase from bovine heart by biomimetic-dye affinity chromatography

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD⁺/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300?u/mg (25?°C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200?mM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD⁺/sulphite elution) and a second ion-exchange chromatography step (elution with 200?mM KCl). The LDH preparation exhibited specific activity approx. 500?u/mg at 25?°C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).     

Selective inhibition of L-glutamate and gammaaminobutyrate transport in nerve ending particles by aluminium,manganese, and cadmium chloride

AlCl₃, MnCl₂, and CdCl₂ inhibited the rates of accumulation of ¹⁴C] L-glutamate and ³H] gammaaminobutyrate (GABA) in purified rat forebrain nerve-ending particles in a dose-dependent fashion. The concentrations that would give 50% inhibition (IC₅₀) of GABA transport were 316 μM, 7.4 mM, and 1.4 mM, respectively. Ca²⁺ (1 mM) enhanced the inhibitory effect of Al³⁺ (IC₅₀ decreased to 149 μM) but antagonized that of Mn²⁺ (IC₅₀ = 10 mM) and Cd²⁺ (IC₅₀ = 2.1 mM). For glutamate transport 1 mM Ca²⁺ changed the IC₅₀ values from 299 to 224 μm for Al³⁺, 7.1 to 10 mM for Mn²⁺, and 2 to 3 mM for Cd²⁺. In contrast, the rates of accumulation of ¹⁴C] 2-deoxy-glucose and ³H] L-phenylalanine were mostly unaffected by these metal ions. The results indicate that Al³⁺, Mn²⁺, and Cd²⁺ exerted selective and differential effects on the transport systems of neurotransmitter substances in the synaptosomal membrane.     

Inhibition of purified bovine liver glutathione reductase with some metal ions

Glutathione reductase (GR; E.C. 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). In this study we tested the effects of Al³⁺, Ba²⁺, Ca²⁺, Li⁺, Mn²⁺, Mo⁶⁺, Cd²⁺, Ni²⁺, and Zn²⁺ on purified bovine liver GR. In a range of 10?μM–10?mM concentrations, Al³⁺, Ba²⁺, Li⁺, Mn²⁺, and Mo⁶⁺, and Ca²⁺ at 5?μM–1.25?mM, had no effect on bovine liver GR. Cadmium (Cd²⁺), nickel (Ni²⁺), and zinc (Zn²⁺) showed inhibitory effects on this enzyme. The obtained IC₅₀ values of Cd²⁺, Ni²⁺, and Zn²⁺ were 0.08, 0.8, and 1?mM, respectively. Cd²⁺ inhibition was non-competitive with respect to both GSSG (Ki_GSSG 0.221?±?0.02?mM) and NADPH (Ki_NADPH 0.113?±?0.008?mM). Ni²⁺ inhibition was non-competitive with respect to GSSG (Ki_GSSG 0.313?±?0.01?mM) and uncompetitive with respect to NADPH (Ki_NADPH 0.932?±?0.03?mM). The effect of Zn²⁺ on GR activity was consistent with a non-competitive inhibition pattern when the varied substrates were GSSG (Ki_GSSG 0.320?±?0.018?mM) and NADPH (Ki_NADPH 0.761?±?0.04?mM), respectively.     

Effects of Cd2+, Mn2+, and Al3+ on rat brain synaptosomal uptake of noradrenaline and serotonin

Cd²⁺, Mn²⁺, and Al³⁺ inhibited synaptosomal amine uptake in a concentration-dependent and time-dependent manner. In the absence of Ca²⁺, the rank order of inhibition of noradrenaline uptake was: Cd²⁺ (IC₅₀ = 250 μM) > Al³⁺ (IC₅₀ = 430 μM) > Mn²⁺ (IC₅₀ = 1.50 mM), the IC₅₀ being the concentration of metal ions that gave rise to 50% inhibition of uptake. In the presence of 1 mM Ca²⁺, the rank order of inhibition of uptake was: Al³⁺ (IC₅₀ = 330 μM) > Cd²⁺ (IC₅₀ = 540 μM) > (IC₅₀ = 1.5 mM). The rank order of inhibition of serotonin uptake without Ca²⁺ was: Al³⁺ (IC₅₀ = 370 μM) > Cd²⁺ (IC₅₀ = 610 μM) > Mn²⁺ (IC₅₀ = 3.4 mM) and the rank order in the presence of 1 mM Ca²⁺ was: Al³⁺ (IC₅₀ = 290 μM) > Cd²⁺ (IC₅₀ = 1.5 mM) > Mn²⁺ (IC₅₀ = 4.0 mM). Ca²⁺, at 1 mM, definitely antagonized the inhibitory actions of Cd²⁺ on noradrenaline and serotonin uptake. Al³⁺ stimulated noradrenaline uptake at concentrations around 20–250 μM but inhibited this uptake at concentrations exceeding 300 μM in a dose-related fashion. Ca²⁺, at 1 mM, enhanced both the stimulatory and inhibitory effects of Al³⁺. Ca²⁺ also enhanced the inhibitory actions of Al³⁺ on seotonin uptake. These results, in conjunction with those we have previously published, suggest that Cd²⁺, Mn²⁺, and Al³⁺ exert differential and selective effects on the structure and function of synaptosomal membranes.     

Accumulation of cadmium in pancreatic β cells is similar to that of calcium in being stimulated by both glucose and high potassium

The transport of Cd²⁺ and the effects of this ion on secretory activity and metabolism were investigated in β cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 μmol/kg dry wt. After 60 min of incubation in a Ca²⁺-deficient medium containing 2.5 μM Cd²⁺ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca²⁺. The incorporation of Cd²⁺ was stimulated either by raising the concentration of glucose to 20 mM or K⁺ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K⁺. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd²⁺-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd²⁺ efflux into a Ca²⁺-deficient medium. Concentrations of Cd²⁺ up to 2.5 μM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd²⁺ concentration was above 10 μM. Basal insulin release was stimulated by 5 μM Cd²⁺. At a concentration of 160 μM, Cd²⁺ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the β cell uptake of Cd²⁺ is facilitated by the activation of voltage-dependent Ca²⁺ channels. Apparently, the accumulation of Cd²⁺ mimics that of Ca²⁺ also involving a component of intracellular sequestration promoted by glucose.     

Effects of epididymal maturation, zinc (II) and copper (II) on the reactive sulfhydryl content of structural elements in rat spermatozoa

The sulfhydryl content of rat spermatozoa at different stages of their maturation in the epididymis was determined by alkylation with ¹⁴C-iodoacetamide. Inhibition of this reaction by reagents having an affinity for thiols verified its specificity. The results support previous conclusions that epididymal maturation in eutherian mammals involves oxidation of -SH groups to -S-S- crosslinks in sperm heads and tails, imparting unusual stability to these structures. The heads and tails of immature rat spermatozoa displayed more than 20 and 5 times as many reactive -SH groups respectively as did those of mature spermatozoa. Fractionation of sonicated spermatozoa revealed that most of the reactive thiols are in the tails. Zn²⁺, Cu²⁺ and Cd²⁺ inhibited the alkylation of -SH groups by iodoacetamide. Although the Zn²⁺ inhibition could be reversed by EDTA, the effect of Cu²⁺, believed to involve oxidation, was not reversible and could be largely prevented by a sufficient excess of Zn²⁺. Thus, Zn²⁺ may retard the oxidation of sperm -SH groups in vivo.     

重金属镉(Cd)在植物体内的转运途径及其调控机制

重金属镉(Cd)的毒害效应与其由土壤向植物地上部分运输有关,揭示Cd~(2+)转运途径及其调控机制可为提高植物抗镉性以及镉污染的植物修复提供依据。对Cd~(2+)在植物体内的转运途径,特别是限制Cd~(2+)移动的细胞结构和分子调控机制研究进展进行了回顾。Cd~(2+)通过共质体和质外体途径穿过根部皮层进入木质部的过程中,大部分在皮层细胞间沉积,少部分抵达中柱后转移到地上部分。为了免受Cd~(2+)的危害,植物体产生了多种限制Cd~(2+)吸收和转移的生理生化机制:1)环绕在内皮层径向壁和横向壁上的凯氏带阻止Cd~(2+)以质外体途径进入木质部;2)螯合剂与进入根的Cd~(2+)螯合形成稳定化合物并区隔在液泡中;3)通过H+/Cd~(2+)离子通道等将Cd~(2+)逆向转运出根部。植物共质体和质外体途径转运重金属镉的能力以及两条途径的串扰尚待进一步明晰和阐明。     

Characterization of human cholesterol side chain cleavage enzyme (EC 1.14.15x) of human term placental mitochondria

The cholesterol side chain cleavage enzyme (EC 1.14.15x) in mitochondria of a human term placenta was partially characterized. Enzyme activity was determined by separation of [26-¹⁴C]-cholesterol and [5-¹⁴C]-isocaproic acid formed by side chain cleavage. Since the amounts of unlabeled cholesterol were too large, a K_M of cholesterol could not be determined. The apparent K_M value of NADPH is 6.25 × 10⁻⁴ M. A pH optimum was found at pH 9.5 (Tris-buffer) and a temperature optimum at 40°C. The metal ions Sr²⁺ and Ba²⁺ showed no inhibition at 1 and 10 mM and a moderate inhibition at 100 mM. In low concentrations (1 mM), Mg²⁺ and Ca²⁺ slightly stimulated the enzyme whereas in higher concentrations (100 mM) an inhibitory effect was observed. A strong inhibition was achieved with 1 mM Zn²⁺, Cd²⁺, Cu²⁺ and by 10 and 100 mM Fe²⁺, Mn²⁺, Co²⁺ and Ni²⁺. During preincubation of the enzyme without radioactive substrate, a rapid loss in enzyme activity in relation to enzyme concentration was observed (initial activity = 100%) (preincubation time in hours): 0.5 h (97%), 1 h (55%) and 1.5 h (34%). A dose-dependent inhibition of the enzyme by the following proteins was achieved: bovine serum protein, human serum protein, human immunoglobulin G and ovalbumin. Furthermore, a dose-dependent inhibition was found with the membrane lipids lecithin and sphingosine.     

Effects of heavy metal cations on the mitochondrial ornithine/citrulline transporter reconstituted in liposomes

The effect of heavy metal cations on the mitochondrial ornithine/citrulline transporter was tested in proteoliposomes reconstituted with the protein purified from rat liver. The transport activity was measured as [³H]ornithine uptake in proteoliposomes containing internal ornithine (ornithine/ornithine antiport mode) or as [³H]ornithine efflux in the absence of external substrate (ornithine/H⁺ transport mode). 0.1 mM Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺ and Zn²⁺ strongly inhibited (more than 85%) the antiport; whereas Mn²⁺, Co²⁺ and Ni²⁺ inhibited less efficiently (25, 47 and 69%, respectively). The IC₅₀ values of the transporter for the different metal ions ranged from 0.71 to 350 μM. Co²⁺ and Ni²⁺ also inhibited the [³H]ornithine efflux whereas Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺ and Zn²⁺ stimulated the [³H]ornithine efflux. The stimulation of the [³H]ornithine efflux by Cu²⁺ and Cd²⁺ (as well as by Pb²⁺, Hg²⁺ and Zn²⁺) was not prevented by NEM and was reversed by DTE. These features indicated that the inhibition of the antiport was due to the interaction of the Cu²⁺, Pb²⁺, Hg²⁺, Cd²⁺ and Zn²⁺ with a population of SH groups, of the transporter, responsible for the inhibition of the physiological function; whereas the stimulation of [³H]ornithine efflux was due to the induction of a pore-like function of the transporter caused by interaction of cations with a different population of SH groups. Differently, the inhibition of the ornithine transporter by Ni²⁺, Co²⁺ or Mn²⁺ was caused by interaction with the substrate binding site, as indicated by the competitive or mixed inhibition.     

Resistance acquisition of Thiobacillus thiooxidans upon cadmium and zinc ion addition and formation of cadmium ion-binding and zinc ion-binding proteins exhibiting metallothionein-like properties

Through subcultivations of Thiobacillus thiooxidans WU-79A in autotrophic media in which the concentrations of Cd²⁺ and Zn²⁺ were increased successively, Cd²⁺-resistant (CDR) and Zn²⁺-resistant strains (ZNR) were obtained. The growth of WU-79A was inhibited by the addition of 25 mM Cd²⁺ as well as Zn²⁺. However, CDR and ZNR could grow without any lag phase in media containing 200 mM Cd²⁺ and 250 mM Zn²⁺, respectively. CDR and ZNR were able to grow even in media containing up to 400 mM Cd²⁺ and 600 mM Zn²⁺, respectively, although they exhibited lag phases. CDR could grow in medium containing up to 250 mM Zn²⁺, as could ZNR in medium containing up to 200 mM Cd²⁺. Cd²⁺-binding and Zn²⁺-binding proteins were isolated from CDR and ZNR, respectively, by gel filtration and ion exchange chromatography. The molecular weights of both proteins were estimated to be approximately 13,000 by gel filtration. The fact that there was no strong absorption at 280 nm of the proteins suggested that they had few aromatic amino acids. Broad absorption bands which are typical of mercaptide (metal thiolate) complexes were detected. The properties of the proteins were spectrophotometrically similar to those of metallothionein.     

Mechanism for Cd2+ inhibition of (K++ Mg2+)ATPase activity and K+ (86Rb+) uptake join roots of sugar beet (Beta vulgaris)

Steady state kinetics were used to examine the influence of Cd²⁺ both on K⁺ stimulation of a membrane-bound ATPase from sugar beet roots (Beta vulgaris L. cv. Monohill) and on K⁺(⁸⁶Rb⁺) uptake in intact or excised beet roots. The in vitro effect of Cd²⁺ was studied both on a 12000–25000 g root fraction of the (Na⁺+K⁺+Mg²⁺)ATPase and on the ATPase when further purified by an aqueous polymer two-phase system. The observed data can be summarized as follows: 1) Cd²⁺ at high concentrations (>100 μM) inhibits the MgATPase activity in a competitive way, probably by forming a complex with ATP. 2) Cd²⁺ at concentrations <100 μM inhibits the specific K⁺ activation at both high and low affinity sites for K⁺. The inhibition pattern appears to be the same in the two ATPase preparations of different purity. In the presence of the substrate MgATP, and at K⁺ <5 mM, the inhibition by Cd²⁺ with respect to K⁺ is uncompetitive. In the presence of MgATP and K⁺ >10 μM, the inhibition by Cd²⁺ is competitive. 3) At the low concentrations of K⁺, Cd²⁺ also inhibits the 2,4-dinitrophenol(DNP)-sensitive (metabolic) K⁺(⁸⁶Rb⁺) uptake uncompetitively both in excised roots and in roots of intact plants. 4) The DNP-insensitive (non metabolic) K⁺(⁸⁶Rb⁺) uptake is little influenced by Cd²⁺. As Cd²⁺ inhibits the metabolic uptake of K⁺(⁸⁶Rb⁺) and the K⁺ activation of the ATPase in the same way at low concentrations of K⁺, the same binding site is probably involved. Therefore, under field conditions, when the concentration of K⁺ is low, the presence of Cd²⁺ could be disadvantageous.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Transition metal ions and selenite modulate the methylation of arsenite by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT)

This report demonstrates that transition metal ions and selenite affect the arsenite methylation by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) in vitro. Co²⁺, Mn²⁺, and Zn²⁺ inhibited the arsenite methylation by hAS3MT in a concentration-dependent manner and the kinetics indicated Co²⁺ and Mn²⁺ to be mixed (competitive and non-competitive) inhibitors while Zn²⁺ to be a competitive inhibitor. However, only a high concentration of Fe²⁺ could restrain the methylation. UV-visible, CD and fluorescence spectroscopy were used to study the interactions between the metal ions above and hAS3MT. Further studies showed that neither superoxide anion nor hydrogen peroxide was involved in the transition metal ion or selenite inhibition of hAS3MT activity. The inhibition of arsenite methylating activity of hAS3MT by selenite was reversed by 2 mM DTT (dithiothreitol) but neither by cysteine nor by β-mercaptoethanol. Whereas, besides DTT, cysteine can also prevent the inhibition of hAS3MT activity by Co²⁺, Mn²⁺, and Zn²⁺. Free Cys residues were involved in the interactions of transition metal ions or selenite with hAS3MT. It is proposed that the inhibitory effect of the ions (Co²⁺, Mn²⁺, and Zn²⁺) or selenite on hAS3MT activity might be via the interactions of them with free Cys residues in hAS3MT to form inactive protein adducts.     

Effect of cadmium and nickel on photosynthesis and the enzymes of the photosynthetic carbon reduction cycle in pigeonpea (Cajanus cajan L.)

The effect of Cd²⁺ and Ni²⁺ on the rate of photosynthesis and activities of key enzymes of the photosynthetic carbon reduction cycle was examined in leaves from pigeonpea (Cajanus cajan L., cv. UPAS-120) grown in nitrogen free sand culture. Two different concentrations of Cd²⁺ and Ni²⁺ were applied through the rooting medium at two growth stages. The application of Cd²⁺ and Ni²⁺ (0.5 and 1.0 mM) at an early vegetative stage (30 days after sowing) resulted in about 50% and 32% reduction in net photosynthesis, respectively. However, enzyme activities were decreased to different levels (2–61%) depending upon the enzyme and the concentration of the metal ion.These concentrations (0.5 and 1.0 mM of Cd²⁺ and Ni²⁺) had no effect when applied at a later vegetative stage i.e. 70 days after sowing. However, when the concentration of Cd²⁺ was increased to 10 mM, there was about an 86% reduction in the rate of photosynthesis but the enzyme activities were reduced by only about 40%. Although Ni²⁺ reduced the photosynthetic rate by 65%, it had little effect on enzyme activities. The reduction in photosynthesis seems to occur indirectly through a decrease in chlorophyll content and stomatal conductance but not due to decreased enzyme activities. Oxygen evolution by leaf discs was inhibited by Cd²⁺ and Ni²⁺ in parallel with a reduction in photosynthesis. These data confirm the earlier reported effects of Cd²⁺ and Ni²⁺ on O₂ evolution in isolated chloroplasts.Abbreviations FBPase Fructose-1,6-bisphosphatase - PCR Photosynthetic carbon reduction - 3-PGA 3-Phospho-glycric acid - RUBP Ribulose, 1,5-bisphosphate     

Inhibition Kinetics of Sheep Brain Glutathione Reductase by Cadmium Ion

Glutathione reductase participates in preventing lipid peroxidation by oxygen radicals which results in cellular damage. The brain is among the organs most susceptible to cadmium-induced lipid peroxidation. The mechanism of free radical generation by Cd²⁺ is not well understood, but it is known that Cd²⁺ is an inhibitor of glutathione reductase. In this study, inhibition kinetics of the brain glutathione reductase by Cd²⁺ was investigated. Sheep brain enzyme (11,000-fold purified) was used for this purpose. The data were analyzed by a nonlinear curve fitting program. It was found that the inhibition was competitive with respect to oxidized glutathione and uncompetitive with respect to NADPH. Inhibition constants were found as 12.3 and 9.4 μM, respectively. These findings might contribute to the understanding of the mechanism of lipid peroxidation by Cd²⁺ in brain.     

Contributions of cell wall and metal-binding peptide to Cd- and Cu-tolerances in suspension-cultured cells of tomato

Possible roles of cell wall and cytoplasmic peptides in the tolerance of cells to Cu²⁺ and Cd²⁺ ions were studied in suspension-cultured cells of tomato (Lycopersicon esculentum L. cv. Palace). Cu²⁺ and Cd²⁺ ions inhibited growth of wild type cells at concentrations more than 100 and 200 μM, respectively. Tomato cells readily developed tolerance to Cd²⁺ ions up to 1 mM but not to Cu²⁺ ions, after repeated subculturings in the presence of the respective ions. Such a metal-specific adaptation of cells was not due to the difference in the total uptakes between Cd²⁺ and Cu²⁺ ions by cells. Wild-type cells accumulated Cd²⁺ preferentially into the cytoplasmic peptide fraction and Cu²⁺ into the cell-wall fraction, when grown under the subtoxic metal conditions. Under excess metal conditions, Cd-tolerant cells produced greater amounts of Cd-binding peptides in the cytoplasm and retained lesser amounts of Cd²⁺ ions in the cell wall than did wild-type cells. In contrast, tomato cells grown in the presence of Cu²⁺ ions synthesized no detectable amounts of Cu-binding peptides in the cytoplasm and retained most of the Cu²⁺ in the cell-wall fraction, irrespective of cell lines. These results suggested that the cytoplasmic peptides rather than cell wall properties have a primary role in the response of tomato cells to excess metal environments.     

Effect of Cadmium Ion on alpha-Glucosidase: An Inhibition Kinetics and Molecular Dynamics Simulation Integration Study

α-Glucosidase is a critical metabolic enzyme that produces glucose molecules by catalyzing carbohydrates. The aim of this study is to elucidate biological toxicity of Cd²⁺ based on α-glucosidase activity and conformational changes. We studied Cd²⁺-mediated inactivation as well as conformational modulation of α-glucosidase by using kinetics coupled with simulation of molecular dynamics. The enzyme was significantly inactivated by Cd²⁺ in a reversibly binding behavior, and Cd²⁺ binding induced a non-competitive type of inhibition reaction (the K _i was calculated as 0.3863 ± 0.033 mM). Cd²⁺ also modulated regional denaturation of the active site pocket as well as overall partial tertiary structural change. In computational simulations using molecular dynamics, simulated introduction of Cd²⁺ induced in a depletion of secondary structure by docking Cd²⁺ near the saccharides degradation at the active site, suggesting that Cd²⁺ modulating enzyme denaturation. The present study elucidated that the binding of Cd²⁺ triggers conformational changes of α-glucosidase as well as inactivates catalytic function, and thus suggests an explanation of the deleterious effects of Cd²⁺ on α-glucosidase.     

Bivalent Metal Ions Modulate Cd2+ Effects on Isolated Rat Liver Mitochondria

We have studied Cd²⁺-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd²⁺] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 M, Cd²⁺ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 M, Cd²⁺ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO₃ or NH₄NO₃ medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd²⁺ effects on mitochondria treated in sequence with 100 M of Ca²⁺, Sr²⁺, Mn²⁺ or Ba²⁺(Me²⁺) and 7.5 M RR, as well as the alterations in Cd²⁺ action on the uptake of ¹³⁷Cs⁺ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca²⁺ or RR. The evidence obtained indicate that Ca²⁺ exhibits a synergestic action on all Cd²⁺ effects examined, whereas Sr²⁺ and Mn²⁺, conversely, are antagonistic. In the presence of RR, the Cd²⁺ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd²⁺, conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd²⁺-stimulated swelling and ¹³⁷Cs⁺ (analog of K⁺) uptake in the acetate media. For the first time, we have shown that Cd²⁺-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.     

Involvement of Ca2+-calmodulin in Cd2+ toxicity during the early phases of radish (Raphanus sativus L.) seed germination

The toxicity of Cd²⁺in vivo during the early phases of radish (Raphanus sativus L.) seed germination and the in vitro Cd²⁺ effect on radish calmodulin (CaM) were studied. Cd²⁺ was taken up in the embryo axes of radish seeds; the increase in fresh weight of embryo axes after 24 h of incubation was inhibited significantly in the presence of 10 mmol m^?3 Cd²⁺ in the external medium, when the Cd²⁺ content in the embryo axes was c. 1.1 μmol g^?1 FW. The reabsorption of K⁺, which characterizes germination, was inhibited by Cd²⁺, suggesting that Cd²⁺ affected metabolic reactivation. The slight effect of Cd²⁺ on the transmembrane electric potential of the cortical cells of the embryo axes excluded a generalized toxicity of Cd²⁺ at the plasma membrane level. After 24 h of incubation, Cd²⁺ induced no increase in total acid-soluble thiols and Cd²⁺-binding peptides able to reduce Cd²⁺ toxicity. Ca²⁺ added to the incubation medium partially reversed the Cd²⁺-induced inhibition of the increase in fresh weight of embryo axes and concomitantly reduced Cd²⁺ uptake. Equilibrium dialysis experiments indicated that Cd²⁺ bound to CaM and competed with Ca²⁺ in this binding. Cd²⁺ inhibited the activation of Ca²⁺-CaM-dependent calf-brain phosphodiesterase, inhibiting the Ca²⁺-CaM active complex. Cd²⁺ reduced the binding of CaM to the Ca²⁺-CaM binding enzymes present in the soluble fraction of the embryo axes of radish seeds. The possibility that Cd²⁺ toxicity in radish seed germination is mediated by the action of Cd²⁺ on Ca²⁺-CaM is discussed in relation to the in vivo and in vitro effects of Cd²⁺.