Correlation of the physicochemical properties of metal ions with their activation and inhibition of human erythrocytic δ-aminolevulinic acid dehydratase (ALAD) in vitro

Using normal adult human whole blood hemolysates this study determined, in a dose-response fashion, the in vitro effects of Na⁺, Mg²⁺, Al³⁺, Mn²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺, Ga³⁺, In³⁺, Sn²⁺, Sn⁴⁺, and Pb²⁺ on normal erythrocytic ALAD. The effects of these 14 metal ions on erythrocytic ALAD 50% inhibited by Pb²⁺ were also determined as was the ability of a maximum stimulatory concentration of Zn²⁺ to prevent or reverse the effects of these metal ions on erythrocytic ALAD. The effects of these metal ions were then classified in terms of their oxidation state, characteristic coordination number, coordination geometry, and hard and soft Lewis acid characteristics in order to determine the physical and chemical properties associated with the ability of a metal ion to activate or inhibit erythrocytic ALAD and whether these properties are unique to a single metal ion. Preincubation studies established Zn²⁺ to be the only metal ion to both activate erythrocytic ALAD and to prevent or reverse the Pb²⁺-induced inhibition of erythrocytic ALAD in vitro even after prolonged contact with the enzyme. Therefore future investigations of the use of nontoxic salts of Zn²⁺ as a prophylactic agent or therapeutic adjunct in the prevention or treatment of lead poisoning with its possibly toxic accumulation of ALA are proposed.     

Mg(II)-induced binding of factor IX-binding protein from the venom of Agkistrodon Halys Pallas with factor Xa

Factor IX-binding protein (AHP IX-bp), a Ca²⁺- and Zn²⁺-binding protein from the venom of Agkistrodon Halys Pallas was reported to bind specifically with factor IX in a Zn²⁺-dependent manner. Here we have purified AHP IX-bp by a simple two-step of chromatography procedure and found that AHP IX-bp also binds factor Xa (FXa) with high binding-affinity in a Mg²⁺-dependent manner. Although Mg²⁺ ions have a significantly low binding-affinity for apo-AHP IX-bp as determined by isothermal titration calorimetry, they can induce the binding of apo-AHP IX-bp with FXa even in the absence of Ca²⁺ as determined by native PAGE and surface plasmon resonance. Mg²⁺ ions are required to maintain in vivo function of FX Gla domain for its recognition of AHP IX-bp. Both Ca²⁺ and Zn²⁺ ions fail to induce the binding between apo-AHP IX-bp and FXa. The abundant Mg²⁺ ions in blood play an important role in the anticoagulation of AHP IX-bp.     

The Formation of Oxytocin Dimers is Suppressed by the Zinc-Aspartate-Oxytocin Complex

The aim of this study was to investigate the effect of divalent metal ions (Ca, Mg^2 +, and Zn^2 +) on the stability of oxytocin in aspartate buffer (pH 4.5) and to determine their interaction with the peptide in aqueous solution. Reversed-phase high-performance liquid chromatography and high-performance size-exclusion chromatography measurements indicated that after 4 weeks of storage at 55°C, all tested divalent metal ions improved the stability of oxytocin in aspartate-buffered solutions (pH 4.5). However, the stabilizing effects ofZn^2 + were by far superior compared with Ca^2 + and Mg^2 +. Liquid chromatography-tandem mass spectrometry showed that the combination of aspartate and Zn^2 + in particular suppressed the formation of peptide dimers. As shown by isothermal titration calorimetry, Zn^2 + interacted with oxytocin in the presence of aspartate buffer, whereas Ca^2 + or Mg^2 + did not. In conclusion, the stability of oxytocin in the aspartate-buffered solution is strongly improved in the presence of Zn^2 +, and the stabilization effect is correlated with the ability of the divalent metal ions in aspartate buffer to interact with oxytocin. The reported results are discussed in relation to the possible mode of interactions among the peptide, Zn^2 +, and buffer components leading to the observed stabilization effects.     

Ferritin: Protection of enzymatic activity against the inhibition by divalent metal ions in vitro

Ferritin binds a large quantity of Be²⁺ (Price D.J. and Joshi, J.G. J. Biol. Chem. 258 (1983) 10873) as well as other divalent metal ions. Therefore the ability of this protein to protect enzymes against or reverse the inhibition by metal ions was studied. Evidence presented here shows that the inhibition by Be²⁺ of the enzymes Na⁺K⁺ATPase, alkaline phosphatase and phosphoglucomutase] is reversed by ferritin. Be²⁺ can be transfered reversibly between phosphoglucomutase and ferritin depending upon the relative concentrations of the 2 proteins. Ferritin also reactivated phosphoglucomutase inhibited by Zn²⁺, Cu²⁺, or Cd²⁺. Incubation of ferritin containing Be²⁺ with 4–10-fold molar excess of phosphoglucomutase (with respect to Be²⁺) removed 90% of the Be²⁺ from ferritin. The rates of inactivation of phosphoglucomutase by Be²⁺ donated by apoferritin or ferritin were identical. Based upon these observations it is suggested that Be²⁺ bound to the protein shell and to the iron core are in equilibrium with each other with the equilibrium favoring ferritin-Be²⁺ complex.     

Zn2+, derived from cell preparation,partly attenuates Ca2+-dependent cell death induced by A23187, calcium ionophore,in rat thymocytes

A23187, a calcium ionophore, is used to induce Ca²⁺-dependent cell death by increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn²⁺ and Fe²⁺ rather than Ca²⁺, trace metal cations in cell suspension may affect Ca²⁺-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1 mM EDTA while it was augmented by 50 μM EDTA and 10 μM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for [Ca²⁺]_i, was significantly reduced by 1 mM EDTA while it was not the case for 50 μM EDTA and 10 μM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for [Zn²⁺]_i, increased by A23187 was respectively reduced by 50 μM EDTA and 10 μM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1 mM EDTA is due to the chelation of extracellular Ca²⁺ and Zn²⁺ while the augmentation by 50 μM ETDA or 10 μM TPEN is due to the chelation of extracellular Zn²⁺. The Tyrode’s solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn²⁺, derived from cell preparation, partly attenuates the Ca²⁺-dependent cell death induced by A23187.     

Effects of cadmium on Drosophila: toxicity, proteins, and transfer RNAs

An animal model with well-defined genetic and biochemical characteristics is needed for a detailed understanding of the mechanism of toxicity by metal ions. Drosophila melanogaster was used in the present study to demonstrate a number of responses of Cd²⁺, including lethality, age-related changes in resistance, alterations of the normal developmental changes in proteins, and alterations in specific transfer RNAs. Genotype-specific differences in resistance to Cd²⁺ were found: the ν; bw strain was 5–10 times more resistant than su(s)²ν; bw for developmental exposure; upon treatment of the young adults the differences were in the same direction, but the sensitivities differed by only two- to three-fold. The adult fly became more sensitive to Cd²⁺ as it aged through 2 weeks, but changed little thereafter. The electrophoretic patterns of proteins of adult flies underwent changes during aging from 1 to 8 days; these changes were markedly altered by 0.55 mm CdCl₂ but not by 0.74 mm ZnCl₂ in the medium on which the flies were maintained. The appearance of queuosine-containing tRNA was stimulated by CdCl₂ (0.05–0.8 mm) in the growth medium, but not by ZnCl₂ (0.07–1.1 mm). Further studies involving D. melanogaster should be useful in defining specific interactions of toxic metal ions with macromolecules to enhance the understanding of the toxic effects of these and similar pollutants.     

Effects of Divalent Cations on Adenosine Agonist Binding to A1 Receptors and Non-A1/Non-A2 Sites in Rat Cerebral Cortex

Abstract: In the present study results are reported concerning the effects of several divalent cations on the binding characteristics of [³H]-cyclohexyladenosine on A1 adenosine receptors and of [³H]-N-ethylcarboxamidoadenosine on non-A1/non-A2 sites in membranes from cerebral cortex of the rat. The [³H]-cyclohexyladenosine binding to A1 receptors was dose-dependently increased by Mn²⁺, Co²⁺, Ca²⁺. The binding characteristics of the agonist were differently affected by Ca²⁺/Mn²⁺ and Mg²⁺. Ca² and Mn²⁺ increased the B_max value without any change in K_d, whereas Mg²⁺ decreased the K_d value without changing the B_max. In the presence of Ca²⁺ and Mg²⁺ the K_d value was similar to that obtained in the presence of Mg²⁺, whereas the B_max value was similar to the apparent number of binding sites calculated in the presence of Ca²⁺. The cations, Cu²⁺, Cd²⁺, Zn²⁺, decreased the Al binding with IC₅₀, values of 19.6 μM, 39.2 μM and 103.9 μM, respectively. The binding characteristics of [³H]-N-ethylcarboxamidoadenosine to non-A1/non-A2 sites were affected by Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺ in the opposite manner to A1 receptors. They decreased the binding with IC₅₀ values of 20.1 mM, 22.8 mM, 93.0 mM and 18.1 mM, respectively. This occurs through an enhancement in K_d, values without changes in the number of binding sites. The findings on A1 receptor and non-A1/non-A2 binding site, taken together, suggest that cations could also exert a modulatory action via specific interactions with divalent cation binding sites on the receptor molecule.     

Effect of CH3HgCl and several transition metals on the dopamine neuronal carrier; peculiar behaviour of Zn2+

CH₃Hg⁺ and metal ions inhibited the specific binding of (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-³H]propenyl) piperazine) ([³H]GBR 12783) to the dopamine neuronal carrier present in membranes from rat striatum with a general rank order of potency CH₃Hg⁺ > Cu²⁺ > Cd²⁺ > Zn²⁺ > Ni²⁺ = Mn²⁺ = Co²⁺, suggesting that -SH groups are chiefly involved in this inhibition. Five millimolar dithiothreitol reversed the rather stable block of the specific binding produced by Cd²⁺ or Zn²⁺. An increase in the concentration of Na⁺, or addition of either K⁺ or Ca²⁺ reduced the inhibitory effects of metal cations, except Cu²⁺. Zn²⁺ (3 μM) reduced the inhibitory potency of Cd²⁺ on the binding but was ineffective against CH₃Hg⁺ and Cu²⁺. Zn²⁺ at 0.3 to 10 μM significantly enhanced the specific binding of [³H]GBR 12783 and [³H]cocaine by 42 to 146%. Zn²⁺ (3 μM) increased the affinity of all pure uptake inhibitors tested and of the majority of the substrates for the [³H]GBR 12783 binding site. Dissociation experiments revealed that Zn²⁺ both inhibited and enhanced the [³H]GBR 12783 binding by recognizing amino acids located close to or in the radioligand binding site. Micromolar concentrations of Zn²⁺ noncompetitively blocked the [³H]dopamine uptake but they did not modify the block of the transport provoked by pure uptake inhibitors. These findings suggest that Na⁺, K⁺, Ca²⁺ and metal ions could recognize some -SH groups located in the [³H]GBR 12783 binding site; low concentrations of Zn²⁺ could allow a protection of these -SH groups.     

Binding of cadmium ions by rat liver and kidney

Most of the bound Cd²⁺, which accumulates in the livers and kidneys of adult rats after the subcutaneous injection of CdCl₂ (2·2 μmoles/100 g body wt) is recovered as a single, heat-stable fraction from the soluble components of these tissues. Although this fraction in either organ also binds Zn²⁺, Cd²⁺ does not displace Zn²⁺ from any of the normal, soluble Zn²⁺-metalloproteins.     

Engineering a high-affinity allosteric binding site for divalent cations in kainate receptors

Kainate receptors are allosterically regulated by sodium ions. Removal of Na⁺ from the extracellular solution, or replacement of Na⁺ by larger monovalent cations, inhibits kainate receptor activity. Sodium binds at a negatively charged cavity in the extracellular neurotransmitter binding domain that is capped by a small hydrophobic residue. Prior work revealed that introduction of aspartic acid at this site strongly reduces GluK2 sensitivity to monovalent cations of different size. We found that the GluK2 M739D mutant is also insensitive to substitution of Na⁺ by the large organic cations Tris and NMDG. Because these are excluded from the Na⁺ binding site by steric hindrance, we investigated the possibility that divalent cations can substitute for Na⁺. We show that in Na⁺ free solutions with low concentrations of Ca²⁺ and Mg²⁺ the GluK2 M739D mutant is inhibited by EDTA; that divalent ions in the micromolar concentration range act as positive allosteric modulators; and that the chemistry of the mutant cation binding site is typical of Ca²⁺ and Mg²⁺ binding sites found in protein crystal structures. Hence, the apparent insensitivity of the M739D mutant to monovalent cations is due to the adventitious allosteric effects of divalent ions at physiological concentrations and below.     

Expression of metallothionein of freshwater crab (Sinopotamon henanense) in Escherichia coli enhances tolerance and accumulation of zinc, copper and cadmium

Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab (Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn²⁺, Cu²⁺ and Cd²⁺ in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins.     

In Vitro Methylation of Arsenite by Rabbit Liver Cytosol: Effect of Metal Ions,Metal Chelating Agents,Methyltransferase Inhibitors and Uremic Toxins

ABSTRACT

The methylation of carrier-free ⁷⁴As-arsenite by liver cytosol of Flemish Giant rabbits is highly susceptible to additions of trace elements. in vitro supplementation of essential trace elements like zinc (Zn²⁺), vanadium (V⁵⁺), iron (Fe²⁺), copper (Cu²⁺) and selenate was shown to increase the methylation efficiency. Trivalent metal ions (e.g. Al³⁺, Cr³⁺ and Fe³⁺), Hg²⁺, Tl⁺ and SeO₃^2- had a deleterious effect. The inhibitory effect of EDTA, oxime and many divalent cations (Ca²⁺, Mg²⁺, Sr²⁺,.) suggest a co-factor role for a specific divalent metal ion, possibly Zn²⁺. Chelating agents used in clinical treatment of acute and chronic inorganic arsenic poisoning lower the methylation capacity of cytosol by rendering the trivalent arsenic unavailable for the methyltransferase enzymes. S-adenosylhomocysteine and periodate-oxidized adenosine, inhibitors of s-adenosylmethionine dependent methylation pathways, inhibit the methylation of arsenite. Pyrogallol, a catechol-O-methyltransferase inhibitor, blocks the action of arsenite- and monomethylarsonic methyltransferase enzymes, suggesting a close structural relationship between the active sites of the different enzymes. Some uraemic toxins, namely oxalate, p-cresol, hypoxanthine, homocysteine and myo-inositol, inhibit arsenic methylation.     

Novobiocin-inhibition and magnesium-interaction of rat liver microsomal bilirubin UDP-glucuronosyltransferase

Novobiocin inhibited bilirubin UDP-glucuronosyltransferase (EC 2.4.1.17) from rat liver both in vitro and in vivo, in a dose-dependent fashion. This inhibition was immediate, and was fully reversed when novobiocin was removed by dialysis or by ultracentrifugation through 0.6 M sucrose. The inhibition could not be explained by an alteration in the membrane conformation of this enzyme, since the same kinetic changes were observed in digitonin-activated and in non-activated microsomes. Novobiocin exerted a non-competitive inhibition of bilirubin UDP-glucuronosyltransferase with either bilirubin or UDP-glucuronic acid as the substrate. Kinetic studies demonstrated uncompetitive inhibition of novobiocin or bilirubin UDP-glucuronosyltransferase as a function of Mg²⁺ concentration, whether the assays were EDTA-free or not. Thus, similarities seem to exist between the known effect of novobiocin on membrane-bound enzymes of the bacterial wall and its inhibitory effect on bilirubin UDP-glucuronosyltransferase: both these enzymic systems require metal divalent cations for maximal activity. The uncompetitive inhibition pattern observed with novobiocin with regard to Mg²⁺ suggests that this antibiotic acts on bilirubin conjugation by affecting Mg²⁺-enzyme complexes.     

Effects od divalent cations on neuromuscular transmission in the chick

In comparative studies on the effects of nine divalent cations (Mn²⁺, Co²⁺, Ni²⁺, Cd²⁺, Zn²⁺, Sr²⁺, Cu²⁺, Ba²⁺ and UO₂²⁺ on twitch responses to indirect and direct stimulations, on acetylcholine response and on Ca²⁺ uptake, it was found that Cd²⁺ was the most potent in inhibiting transmitter release from the motor nerve terminals but was without appreciable effect on the chick biventer cervicis muscle. Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ were less potent than Cd²⁺ in producing inhibition at presynaptic sites but were morep potent at postsynaptic sites. By contrast, UO₂²⁺ potentiated twitch responses to indirect stimulation and induced a contracture of the muscle in the presence of physostigmine. Cu²⁺ and Ba²⁺ were particularly potent in inducing a contracture and at higher concentrations abolished twitch responses to indirect and direct stimulation. Sr²⁺ at the very high concentration of 22 mM had only a week inhibitory action on twitch responses but was the only cation capable of substituting for Ca²⁺ in inducing contraction. The studies on the effects of these cations on denervated muscle also led to the conclusion that Cd²⁺ and UO₂²⁺ act mainly on presynaptic nerve terminals and that Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ act on presynaptic sites preferentially to postsynaptic sites while Cu²⁺ and Ba²⁺ act mainly on muscle. The effects of these cations can be antagonized by high Ca²⁺ (10 mM) but those of Cu²⁺ cannot. Cysteine (2.5p mM) antagonized the effects of all the cations except Mn²⁺, Ba²⁺ and UO₂²⁺. d-Tubocurarine suppressed the contracture induced by UO₂²⁺ but not that induced by Cu²⁺ and Ba²⁺. These cations affected ⁴⁵Ca²⁺ uptake and ionic contents of the muscle differently. ⁴⁵Ca²⁺ uptake of the muscle was decreased by Mn²⁺, Co²⁺ and Ni²⁺, not altered by UO₂²⁺ but increased by Cd²⁺, Zn²⁺, Sr²⁺, Cu²⁺ and Ba²⁺. In addition to greatly increasing ⁴⁵Ca²⁺ uptake, Cu²⁺ also increased calcium and sodium but decreased the potassium content of muscle. Zn²⁺ and Sr²⁺ increasedp the calcium content slightly but the other cations did not effect ionic contents appreciably. It is concluded that these cations exert their effects on nerve or on muscle depending on the ligands of the ligands of the membrane bindingp these cations. Some of the cations bi-nd with the—SH group of the membrane and thus alter the transmembrane movement of Ca²⁺ bu the others do not.     

Studies with tryptophan metabolites in vitro. effect of zinc,manganese, copper and cobalt ions on kynurenine hydrolase and kynurenine aminotransferase in normal mouse liver

The possible mechanisms by which increasing concentrations of Zn²⁺, Mn²⁺, Cu²⁺, or Co²⁺ may affect the vitamin B₆-dependent enzymes kynurenine hydrolase and kynurenine aminotransferase. were studied in normal mouse liver homogenates. It was found that Zn²⁺ inhibited kynurenine hydrolase. whereas Mn²⁺ activated this enzyme, but both Zn²⁺ and Mn²⁺ activated kynurenine aminotransferase. Co²⁺ and Cu²⁺ inhibited both enzymes. This inhibition is attributed to the blocking and inactivation of the -SH groups of these enzymes and may be due to the adequate sequence of the -SH groups in both enzymes for Cu²⁺ or Co²⁺ action. This is in contrast to the inadequate sequence of these groups in kynurenine aminotransferase for Zn²⁺ action. The decreasing order by which these metal ions inhibit (a) kynurenine hydrolase is Cu²⁺ > Co²⁺ > Zn²⁺, and (b) kynurenine aminotransferase is Cu²⁺ > Co²⁺. The decreasing order of the per cent activation of the aminotransferase enzyme is Mn²⁺ > Zn²⁺. These decreasing orders fall into a more reasonable order approximating the order of complex stability of these metal ions.     

The Interaction Between Oxytetracycline and Divalent Metal Ions in Aqueous and Mixed Solvent Systems

The effects of pH, mixed solvent systems, and divalent metal ions on oxytetracycline (OTC) solubility and the interactions between OTC and metal ions in aqueous and mixed solvent systems were investigated. OTC solubility profiles were obtained for pH 4–9. The cosolvents studied were glycerin, propylene glycol, PEG 400, and 2-pyrrolidone with the following metal ions: magnesium, calcium, and zinc. OTC and its interactions with these metal ions were evaluated by solubility, NMR, circular dichroism (CD), and electron diffraction (ED) methods. At pH 5.6, no complexation occurred with these metal ions, but OTC zwitterion formed aggregates in aqueous solutions as shown by NMR spectra. The hydration of the metal ions was observed to affect OTC aggregation, with Mg⁺² causing the greatest OTC aggregation. At pH 7.5, OTC aggregation and metal–OTC complexation were observed in solutions with Ca⁺² and Mg⁺². Zinc ion was found to decrease OTC solubility because of zincate formation, which caused anionic OTC to precipitate. Electron diffraction revealed a relationship between OTC and metal–OTC complex crystallinity and solubility behavior. The zinc–OTC complex exhibited the highest crystallinity and lowest solubility at pH 8.0. Various cosolvents generally enhanced OTC solubility, with 2-pyrrolidone having the best solubility power. In OTC–metal-2-pyrrolidone and OTC–Zn⁺²-PEG 400 systems, circular dichroism provided evidence for the formation of soluble ternary complexes.