Percorrer por autor "Duarte, Rui O."
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- Decavanadate interactions with actin: cysteine oxidation and vanadyl formationPublication . Ramos, Susana; Duarte, Rui O.; Moura, José J. G.; Aureliano, M.Incubation of actin with decavanadate induces cysteine oxidation and oxidovanadium(IV) formation. The studies were performed combining kinetic with spectroscopic (NMR and EPR) methodologies. Although decavanadate is converted to labile oxovanadates, the rate of deoligomerization can be very slow (half-life time of 5.4 h, at 25 ◦C, with a first order kinetics), which effectively allows decavanadate to exist for some time under experimental conditions. It was observed that decavanadate inhibits F-actin-stimulated myosin ATPase activity with an IC50 of 0.8 mMV10 species, whereas 50 mMof vanadate or oxidovanadium(IV) only inhibits enzyme activity up to 25%. Moreover, from these three vanadium forms, only decavanadate induces the oxidation of the so called “fast” cysteines (or exposed cysteine, Cys-374) when the enzyme is in the polymerized and active form, F-actin, with an IC50 of 1 mMV10 species. Decavanadate exposition to F- and G-actin (monomeric form) promotes vanadate reduction since a typical EPR oxidovanadium(IV) spectrum was observed. Upon observation that V10 reduces to oxidovanadium(IV), it is proposed that this cation interacts with G-actin (Kd of 7.48 ± 1.11 mM), and with F-actin (Kd = 43.05 ± 5.34 mM) with 1:1 and 4:1 stoichiometries, respectively, as observed by EPR upon protein titration with oxidovanadium(IV). The interaction of oxidovanadium(IV) with the protein may occur close to the ATP binding site of actin, eventually with lysine-336 and 3 water molecules.
- Decavanadate interactions with actin: inhibition of G-actin polymerization and stabilization of decameric vanadatePublication . Ramos, Susana; Manuel, Miguel; Tiago, Teresa; Duarte, Rui O.; Martins, Jorge; Gutiérrez-Merino, Carlos; Moura, José J. G.; Aureliano, M.Decameric vanadate species (V10) inhibit the rate and the extent of G-actin polymerization with an IC50 of 68 ± 22 lM and 17 ± 2 lM, respectively, whilst they induce F-actin depolymerization at a lower extent. On contrary, no effect on actin polymerization and depolymerization was detected for 2 mM concentration of ‘‘metavanadate’’ solution that contains ortho and metavanadate species, as observed by combining kinetic with 51V NMR spectroscopy studies. Although at 25 C, decameric vanadate (10 lM) is unstable in the assay medium, and decomposes following a first-order kinetic, in the presence of G-actin (up to 8 lM), the half-life increases 5-fold (from 5 to 27 h). However, the addition of ATP (0.2 mM) in the medium not only prevents the inhibition of G-actin polymerization by V10 but it also decreases the half-life of decomposition of decameric vanadate species from 27 to 10 h. Decameric vanadate is also stabilized by the sarcoplasmic reticulum vesicles, which raise the half-life time from 5 to 18 h whereas no effects were observed in the presence of phosphatidylcholine liposomes, myosin or G-actin alone. It is proposed that the ‘‘decavanadate’’ interaction with G-actin, favored by the G-actin polymerization, stabilizes decameric vanadate species and induces inhibition of G-actin polymerization. Decameric vanadate stabilization by cytoskeletal and transmembrane proteins can account, at least in part, for decavanadate toxicity reported in the evaluation of vanadium (V) effects in biological systems.
- Interactions of vanadium(V)–citrate complexes with the sarcoplasmic reticulum calcium pumpPublication . Aureliano, M.; Tiago, Teresa; Gândara, Ricardo M. C.; Sousa, Andrea; Moderno, A.; Kaliva, M.; Salifoglou, A.; Duarte, Rui O.; Moura, José J. G.Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)–citrate complexes, namely (NH4)6[V2O4(C6H4O7)2] Æ 6H2O and (NH4)6[V2O2(O2)2(C6H4O7)2] Æ 4H2O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with 51V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4–0.5 mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1 mM solution of the PVC complex, containing 184 lM of the PVC complex, 94 lM oxoperoxovanadium(V) (PV) species, 222 lM monomeric (V1), 43 lM dimeric (V2) and 53 lM tetrameric (V4) species, inhibits Ca2+ accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98 lM of the VC complex, 263 lM monomeric (V1), 64 lM dimeric (V2) and 92 lM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460 lM monomeric (V1), 90.2 lM dimeric (V2) and 80 lM tetrameric (V4) species, has no effect on Ca2+ accumulation. The NMR signals from the VC complex ( 548.0 ppm), PVC complex ( 551.5 ppm) and PV ( 611.1 ppm) are broadened upon SR vesicle addition (2.5 mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4 > PVC > VC > PV > V2 = V1 = 1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium( V)–citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.
- Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2-6-dicarboxylatodioxovanadium(V), BMOV and an amavadine analoguePublication . Aureliano, M.; Henao, Fernando; Tiago, Teresa; Duarte, Rui O.; Moura, José J. G.; Baruah, Bharat; Crans, Debbie C.The general affinity of the sarcoplasmic reticulum (SR) Ca2+-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis(N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes’ catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca2+-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC50 values were 25, 40, 80, and 325 μM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca2+-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca2+-ATPase.
- Vanadate oligomers interaction with phosphorylated myosiPublication . Aureliano, M.; Tiago, Teresa; Duarte, Rui O.; Moura, José J. G.Using a myosin preparation containing endogenous myosin light-chain (LC2) kinase and phosphatase and calmodulin, i.e. near physiological ones, the interaction of vanadate oligomers with phosphorylated myosin was evaluated. Decavanadate or metavanadate solutions (2/15 mM totalvanadate) did not prevent the phosphorylation state of the regulatory myosin lightchain, as observed by urea-polyacrylamide gel electrophoresis. The relative order of line broadening upon protein addition, reflecting the interaction of thevanadate oligomers with phosphorylated myosin, was V10!/V4!/V 10/1 whereas, no changes were observed for monomericvanadate. In the presence of ATP, V1 signal was shifted upfield 2 ppm and became broadened, while V4 signal became narrowed. Moreover, a significant increase in myosin ATPase inhibition (60%) was observed when decameric vanadate species were present (1.4 mM). It is concluded that, under conditions near physiological ones, decamericvanadate differs fromvanadate oligomers present in metavanadate solutions due to its strong interaction with the phosphorylated enzyme and myosinATPaseinhibition.Besides,ATPdecreasestheaffinityofmyosinfortetravanadate,inducestheinteractionwithmonomeric vanadate, whereas it does not affect decamericvanadate interaction.
- Vanadium distribution, lipid peroxidation and oxidative stress markers upon decavanadate in vivo administrationPublication . S. Soares, Sandra; Martins, H.; Duarte, Rui O.; Moura, José J. G.; Coucelo, Josefina; Gutiérrez-Merino, Carlos; Aureliano, M.The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1 mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12 h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12 h after metavanadate administration, whilst for decavanadate no effects were observed except 1 h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12 h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases ( 55%) mitochondrial CAT activity. At early times of exposure, 1 and 6 h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.