On the interaction of vanadium species with meso-2,3-dimercaptosuccinic acid

The interaction of the VO²⁺ cation with meso-2,3-dimercaptosuccinic acid (DMSA) was investigated by electron absorption spectroscopy in aqueous solution at different pH values. The spectral behavior, complemented with a spectrophotometric titration, shows the generation of a [VO(DMSA)₂]²⁻ complex in which the oxocation interacts with two pairs of deprotonated-SH groups of the acid. It was also found that DMSA rapidly reduces VO₃ ⁻ to VO²⁺, which might be chelated by an excess of the acid. DMSA can also produce the partial reduction of a V₂O₅ suspension at pH=5.2. The results of this study suggest that DMSA might be a potentially useful detoxification agent for vanadium.     

Interaction of the vanadyl (IV) cation with carnosine and related ligands

The interaction of the vanadyl (IV) (VO²⁺) cation with carnosine (the dipeptide β-alanyl-histidine) has been investigated by electron absorption spectroscopy at high ligand-to-metal ratios and at different pH values. The results show that in the range 6.0–8.5, the cation interacts with the imidazole group of four different carnosine molecules and points to the presence of an axially coordinated water molecule. These suppositions were confirmed by the behavior of the VO²⁺/imidazole system, which was investigated under similar experimental conditions, and supported by previous ENDOR (electron-nuclear double resonance) results. The study was complemented with additional measurements using the glycylglycine, glycylglycine/imidazole, and histidine systems as ligands.     

On the Interaction of Vanadium Species with the Monoisoamyl Ester of <Emphasis Type="Italic">Meso</Emphasis>-2,3-Dimercaptosuccinic Acid

The interaction of the VO²⁺ cation with the monoisoamyl ester of meso-2,3-dimercaptosuccinic acid (MiADMSA) was investigated by electron absorption spectroscopy in aqueous solutions at different pH values. The spectral behavior, complemented with a spectrophotometric titration, shows the generation of a [VO(MiADMSA)₂]⁴⁻ complex in which the oxocation interacts with two pairs of deprotonated –SH groups of the ester. Besides, MiADMSA rapidly reduces VO₃⁻ to VO²⁺, which might be chelated by an excess of the ester, and also produces relatively rapid reduction of V₂O₅ suspensions at pH = 6.5. The results of this study suggest that MiADMSA might be a potentially useful detoxification agent for vanadium.     

Vanadyl(IV) conalbumin. II. Mixed metal and anion binding studies

Electron paramagnetic resonance (epr) studies demonstrate that at low levels of conalbumin (CA) saturation with Fe³⁺ or VO²⁺, a ph-dependent preference of the metal exists for different protein binding-site configurations,A, B, and C. The vanadyl ion epr spectra of mixed VO²⁺, Fe³⁺-conalbumin in which Fe³⁺ is preferentially bound to the N- or C-terminal binding site are consistent with all three configurations being formed at both metal sites. At high pH the spectra suggest interaction between binding sites. In the absence of HCO₃^?, VO²⁺ is bound almost exclusively in B configuration; a full binding capacity of 2 VO²⁺ per CA is retained. Stoichiometric amounts of HCO₃^? convert the epr spectrum from B to an A, B, C type. Addition of oxalate to bicarbonate-free preparations converts the B spectrum to an A′, B, C′ type where the B resonances have lost intensity to the A′ and C′ resonances but have not changed position. The data suggest that configuration B is anion independent and that only one equivalent of binding sites at pH 9 responds to the presence of HCO₃^1? or oxalate by changing configuration but not metal binding capability. The form of the bound anion may be HCO₃^? rather than CO₃^2?. The formation rate of the colored ferric conalbumin complex by oxidizing Fe²⁺ to Fe³⁺ in limited HCO₃^? at pH 9 is also consistent with one equivalent of sites having different anion requirements than the remaining sites. Increased NaCl or NaClO₄ concentration or substitution of D₂O for water as solvent affect the environment of bound VO²⁺, but the mechanisms of action are unknown.     

Two galactomannan preparations from seeds from Mimosa scabrella (bracatinga): Complexation with oxovanadium(IV/V) and cytotoxicity on HeLa cells

Two galactomannans, GALMAN-A and GALMAN-B, were isolated from seeds of Mimosa scabrella (bracatinga), with deactivation and exposure to native enzymes, respectively. They were treated with oxovanadium(IV) and oxovanadium(V), designated (VO²⁺/VO³⁺) to form GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ complexes, respectively. The potentiometric studies provided the binding constants for the complexes and the resulting complexed species were a function of pH. ⁵¹V NMR spectra of GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ at pH 7.8 and at 30 °C indicated the occurrence of two types of complexes formed by oxovanadium ions and galactomannans. GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ caused loss of HeLa cells viability at concentrations of 50-200 μg/mL. GALMAN-A:VO²⁺/VO³⁺ exhibited low toxicity for 24 h, although GALMAN-B:VO²⁺/VO³⁺ was extremely toxic, since 50 μg/mL was sufficient to decrease HeLa cell viability after 48 h by 60%. GALMAN-A gave rise to a slight increase in cell proliferation after 48 h at 100 μg/mL, whereas GALMAN-B promoted a slight decrease at concentrations of 50-100 μg/mL. GALMAN-A:VO²⁺/VO³⁺ and GALMAN-B:VO²⁺/VO³⁺ exhibited a significant decrease in cell proliferation after 48 h, each reaching 60% inhibition at 5-10 μg/mL. The complexes which caused this effect were at concentrations 10 times lower than the uncomplexed polymers.     

Interaction of VO ion with human serum transferrin and albumin

The complexation of VO²⁺ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO²⁺ ion occupies three different binding sites, A, B₁ and B₂, distinguishable in the X-band anisotropic spectrum recorded in D₂O. With albumin the results show that a dinuclear complex (VO)₂^dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO²⁺, the multinuclear complex (VO)_x^mHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the ⁵¹V A_z values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO²⁺/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO²⁺ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.     

The interaction of the vanadyl(IV) cation with phytic acid

The interactions of VO²⁺ with phytate to form both soluble and insoluble complexes, have been studied by electronic absorption spectroscopy. A soluble 1∶1 VO²⁺: phytate complex is formed at pH <1. At higher pH-values insoluble complexes are produced. Two different solid complexes, obtained respectively at pH=2 and 4, were isolated and characterized. The maximal bonding ratio of VO²⁺: phytate was found to be 4, on the basis of a pH binding profile.     

Characterization of a novel vanadium-binding protein (VBP-129) from blood plasma of the vanadium-rich ascidian Ascidia sydneiensis samea

The ascidians, the so-called sea squirts, accumulate high levels of vanadium, a transition metal. Since Henze first observed this physiologically unusual phenomenon about one hundred years ago, it has attracted interdisciplinary attention from chemists, physiologists, and biochemists. The maximum concentration of vanadium in ascidians can reach 350 mM, and most of the vanadium ions are stored in the + 3 oxidation state in the vacuoles of vanadium-accumulating blood cells known as vanadocytes. Many proteins involved in the accumulation and reduction of vanadium in the vanadocytes, blood plasma, and digestive tract have been identified. However, the process by which vanadium is taken in prior to its accumulation in vanadocytes has not been elucidated. In the present study, a novel vanadium-binding protein, designated VBP-129, was identified from blood plasma of the vanadium-rich ascidian Ascidia sydneiensis samea. Although VBP-129 mRNA was transcribed in all A. sydneiensis samea tissues examined, the VBP-129 protein was exclusively localized in blood plasma and muscle cells of this ascidian. It bound not only to VO²⁺ but also to Fe³⁺, Co²⁺, Cu²⁺, and Zn²⁺; on the other hand, a truncated form of VBP-129, designated VBP-88, bound only to Co²⁺, Cu²⁺ and Zn²⁺. In a pull-down assay, an interaction between VanabinP and VBP-129 occurred both in the presence and the absence of VO²⁺. These results suggest that VBP-129 and VanabinP function cooperatively as metallochaperones in blood plasma.     

Aminoacid-derivatised picolinato-oxidovanadium(IV) complexes: Characterisation, speciation and ex vivo insulin-mimetic potential

The proligands PicMe-AaR (PicMe = methoxipicolyl-5-amide, where the amide substituent is an amino acid AaR = HisH, HisMe, IleH, IleMe, TrpH, TrpMe, HTyrEt, tBuTyrMe, HThrMe, tBuThrMe) and the complexes [VO(Pic-AaR)₂] have been synthesised and characterised. A detailed EPR study of the VO²⁺/Pic-His systems in water revealed the predominance of the complex [VO(Pic-His)H₂O] in the pH range 2-6, with tridentate coordination of Pic-His via the picolinate moiety and imidazole-Nδ. Speciation analyses of the binary systems VO²⁺/Pic-Aa (Aa = His, Ile, Trp) and the ternary systems VO²⁺/Pic-Aa/B (Aa = His, Ile; B = citrate (cit), lactate (lac), phosphate) showed a predominance of the ternary complexes [VO(Pic-Aa)(cit/lac)] and [VO(Pic-Aa)(cit/lac)OH]⁻ in the physiological pH regime. If, in addition, human serum albumin (HAS) and apotransferrin (Tf) are present, with all of the low and high molecular mass constituents in their blood serum concentrations, about two thirds of VO²⁺ is bound to the protein, while there is still a sizable amount of ternary complex [VO(Pic-Aa)(cit/lac)] present (about 1/4 for Pic-His and 1/3 for Pic-Ile) when the vanadium(IV) concentration is relatively high; at lower concentrations Tf is the predominant binder. Insulin-mimetic studies for VO²⁺/Pic-Aa (Aa = His, Ile, Tyr and Trp), based on a lipolysis assay with rat adipocytes, provided IC₅₀ values of 0.41(1) for VO²⁺/Pic-His and VO²⁺/Pic-Ile, which compares with 0.87(17) for VOSO₄.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis

Background

Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO²⁺ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined.

Methods

In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method.

Results

Mutation at K10/R60 (site 1) markedly reduced the affinity for VO²⁺. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO²⁺. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO²⁺.

General significance

These results suggested that the site 1 is a high affinity binding site for VO²⁺, while the site 2 composes a moderate affinity site for multiple VO²⁺.     

Influence of the protonation,deprotonation and transition metal ions on the fluorescence of 8-hydroxyquinoline: a computational study

8-Hydroxyquinoline (8HyQ) and its derivatives are the important constituents in a variety of pharmaceutical compounds. The effect of protonation and deprotonation of 8HyQ on its electronic structure and fluorescence was investigated using B3LYP/6-311G** level of theory. We also investigated the interaction of chemosensor, 8HyQ, with different transition metals (Zn²⁺, Fe²⁺, Ni²⁺ and Co²⁺) at the same level. Our results revealed that 8HyQ displays an unusual fluorescence intensity–proton transfer relationship with diminished emission in a protonated form but enhanced emission in a deprotonated form. The Zn²⁺, Fe²⁺, Ni²⁺ and Co²⁺ complexes of 8HyQ, which were investigated at the same level of theory, showed that the order of binding energies was 8HyQ-Ni²⁺>8HyQ-Zn²⁺>8HyQ-Co²⁺>8HyQ-Fe²⁺. Time-dependent density functional theory calculations indicated that Zn ion enhances the fluorescence of 8HyQ as a consequence of the inhibition of the proton transfer. The results are in good agreement between the predicted properties of transition metal complexes of 8HyQ and previously published experimental and theoretical results. A natural bond orbital analysis was performed to understand the nature of hydrogen-bonding interaction in 8HyQ and also to reveal the inter-relations between electronic structure and other properties.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea

Background

Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified.

Methods

We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function.

Results

Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO²⁺ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K_m and V_max) of AsNramp-mediated VO²⁺ transport at pH 8.5 were 90 nM and 9.1 pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO²⁺ under the same conditions. Excess Fe²⁺, Cu²⁺, Mn²⁺, or Zn²⁺ inhibited the transport of VO²⁺. AsNramp was revealed to be a novel VO²⁺/H⁺ antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H⁺-ATPase in vanadocytes.

General Significance

This is the first report of identification and functional analysis on a membrane transporter for vanadium ions.     

Characterization of junctate-SERCA2a interaction in murine cardiomyocyte

Junctate is a newly identified sarcoplasmic reticulum (SR) Ca²⁺ binding protein, but its function in cardiac muscle has remained unclear. Our previous study showed that chronic over-expression of junctate in transgenic mice led to altered SR functions and development of severe hypertrophy. In this study, we identified the interaction of junctate with SERCA2a by co-immunoprecipitation and GST-pull-down assay. This interaction was inhibited by higher Ca²⁺ concentration. Immunolocalization assays also showed that junctate and SERCA2a were co-localized in the SR of cardiomyocytes. Direct binding of the C-terminal region of junctate (amino acids 79-270) and luminal domain of SERCA2a (amino acids 70-89) was observed by deletion mutation experiments. Adenovirus-mediated transient over-expression of junctate in cardiomyocytes showed a reduced decay time of Ca²⁺ transients and increased oxalate-supported SERCA2 Ca²⁺ uptake, suggesting an increased activity of SERCA2a. Taken together, according to our data, junctate may play an important role in the regulation of SR Ca²⁺ cycling through the interaction with SERCA2a in the murine heart.     

One and Two Dimensional Pulsed Electron Paramagnetic Resonance Studies of in vivo Vanadyl Coordination in Rat Kidney

The biological fate of a chelated vanadium source is investigated by/n vivo spectroscopic methods to elucidate the chemical form in which the metal ion is accumulated. A pulsed electron paramagnetic resonance study of vanadyl ions in kidney tissue, taken from rats previously treated with bis(ethylmaltolato)oxovanadium(IV) (BEOV) in drinking water, is presented. A combined approach using stimulated echo (3-pulse) electron spin echo envelope modulation (ESEEM) and the two dimensional 4-pulse hyperfine sublevel correlation (HYSCORE) spectroscopies has shown that at least some of the VO²⁺ ions are involved in the coordination with nitrogen-containing ligands. From the experimental spectra, a 4N hyperfine coupling constant of 4.9 MHz and a quadrupole coupling constant of 0.6 + 0.04 MHz were determined, consistent with amine coordination of the vanadyl ions. Study of VO-histidine model complexes allowed for a determination of the percentage of nitrogen-coordinated VO²⁺ ions in the tissue sample that is found nitrogen-coordinated. By taking into account the bidentate nature of histidine coordination to VO²⁺ ions, a more accurate determination of this value is reported. The biological fate of chelated versus free (i.e. salts) vanadyl ion sources has been deduced by comparison to earlier reports. In contrast to its superior pharmacological efficacy over VOSO4, BEOV shares a remarkably similar biological fate after uptake into kidney tissue.     

Si‐Doping Mediated Phase Control from β‐ to γ‐Form Li3VO4 toward Smoothing Li Insertion/Extraction

The γ phase Li₃VO₄ which possesses higher ionic conductivity is more preferable for lithium ion batteries, but it is only stable at high temperature and would convert to low temperature β phase spontaneously when cooling down. Here, the phase control of Li₃VO₄ to stabilize its γ phase in room temperature is successfully mediated by introducing proper Si‐doping, and for the first time the electrochemical performances of γ‐Li₃VO₄ is investigated. It is found that pure γ‐Li₃VO₄ can be obtained in a doping ratio of x = 0.05–0.15 in Li_3+xV_1?xSi_xO₄ with addition of excess Li source in synthesis design. The doping mechanism and the energy changes are investigated in detail by using the first principle calculations, it reveals that an interstitial Li⁺ is formed with doping of Si⁴⁺ in Li₃VO₄ to balance the system charge. When served as an anode, the Si‐doped γ‐Li₃VO₄ shows much smoothed Li⁺ insertion/extraction and enhanced cycle stability with only a single pair of redox peaks, which behaves much different with the complex multicouples of redox peaks in typical β‐Li₃VO₄. These changes in electrochemical performances implies that γ‐Li₃VO₄ can effectively accommodate Li⁺ in an easier and more facile way and relieved structure stress during the charge/discharge process.     

Metal complexes of N-hydroxy-imino-di-α-propionic acid and related ligands

N-hydroxy-imino-di-α-propionic acid, the ligand present in the natural oxovanadium(IV) complex ‘amavadin’ which occurs in the toadstool Amanita muscaria, has been synthesised, as well as two related ligands—N-hydroxy-iminodiacetic acid and imino-di-α-propionic acid—useful for comparison purposes. The formation of complexes of these ligands with VO²⁺, Ni²⁺ has been studied and their stability constants have been determined.The two N-hydroxy-substituted ligands, of low basicity, form ML₂ complexes with VO²⁺, unlike the more basic derivatives of iminodiacetic acid. Since substitution of ligands bonded to the apical site trans to the oxo ligand is very fast and the formation of ML₂ complexes of VO²⁺ exposes that apical site to the reaction media, this may be the reason why oxovanadium(IV) and the unusual derivative of iminodiacetic acid present in ‘amavadin’ were selected for the biological role that this complex plays in the toadstool.     

Homocysteine and cognitive function in elderly people

DEMENTIA IS HIGHLY PREVALENT AMONG ELDERLY PEOPLE, and projections show that the number of people affected might triple over the next 50 years, mainly because of a large increase in the oldest-old segment of the population. Because of this and the disease''s devastating effects, measures for the prevention and early detection of dementia are crucial. Age and years of education are among the most relevant risk factors for dementia, but in recent years the role of homocysteine has also been investigated. Homocysteine is an amino acid produced in the metabolism of methionine, a process dependent on the B vitamins cobalamin, vitamin B₆ and folic acid. There is evidence that increased serum homocysteine levels are associated with declining cognitive function and dementia. We review this evidence in addition to the potential mechanisms through which homocysteine acts on the brain to cause cognitive dysfunction, the metabolism of homocysteine and factors associated with alteration of the normal metabolism. Dementia is characterized by a progressive deterioration of cognitive skills that leads to a decline in the ability to perform daily activities. It affects 8% of people over the age of 65¹ and results in more than 60 000 new cases in Canada each year. Alzheimer''s disease accounts for more than 50% of cases of dementia in Canada.² Projections for the next 50 years show that the number of patients with dementia might triple,³ mainly because of a large increase in the oldest-old segment of the population. Because of the disease''s high prevalence and devastating effects on patients, caregivers and the health care system, measures for the prevention and early detection of dementia are crucial.Age and years of education are among the most relevant risk factors for dementia, but in recent years the role of homocysteine has also been investigated. Homocysteine is an amino acid that is produced in the metabolism of methionine, a process dependent on the B vitamins cobalamin, vitamin B₆ and folic acid. There is evidence that increased serum homocysteine levels are associated with declining cognitive function and dementia. We review this evidence in addition to the potential mechanisms through which homocysteine acts on the brain to cause cognitive dysfunction, the metabolism of homocysteine and factors associated with alteration of the normal metabolism.