title: Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate.
abstract: Over the last few decades there has been increasing interest in oxometalate and polyoxometalate applications to medicine and pharmacology. This interest arose, at least in part, due to the properties of these classes of compounds as anti-cancer, anti-diabetic agents, and also for treatment of neurodegenerative diseases, among others. However, our understanding of the mechanism of action would be improved if biological models could be used to clarify potential toxicological effects in main cellular processes. Sarcoplasmic reticulum (SR) vesicles, containing a large amount of Ca(2+)-ATPase, an enzyme that accumulates calcium by active transport using ATP, have been suggested as a useful model to study the effects of oxometalates on calcium homeostasis. In the present article, it is shown that decavanadate, decaniobate, vanadate, tungstate and molybdate, all inhibited SR Ca(2+)-ATPase, with the following IC(50) values: 15, 35, 50, 400 μM and 45 mM, respectively. Decaniobate (Nb(10)), is the strongest P-type enzyme inhibitor, after decavanadate (V(10)). Atomic-absorption spectroscopy (AAS) analysis, indicates that decavanadate binds to the protein with a 1:1 decavanadate:Ca(2+)-ATPase stoichiometry. Furthermore, V(10) binds with similar extension to all the protein conformations, which occur during calcium translocation by active transport, namely E1, E1P, E2 and E2P, as analysed by AAS. In contrast, it was confirmed that the binding of monomeric vanadate (H(2)VO(4)(2-); V(1)) to the calcium pump is favoured only for the E2 and E2P conformations of the ATPase, whereas no significant amount of vanadate is bound to the E1 and E1P conformations. Scatchard plot analysis, confirmed a 1:1 ratio for decavanadate-Ca(2+)-ATPase, with a dissociation constant, k(d) of 1 μM(-1). The interaction of decavanadate V(10)O(28)(6-) (V(10)) with Ca(2+)-ATPase is prevented by the isostructural and isoelectronic decaniobate Nb(10)O(28)(6-) (Nb(10)), whereas no significant effects were detected with ATP or with heparin, a known competitive ATP binding molecule, suggesting that V(10) binds non-competitively, with respect to ATP, to the protein. Finally, it was shown that decaniobate inhibits SR Ca(2+)-ATPase activity in a non competitive type of inhibition, with respect to ATP. Taken together, these data demonstrate that decameric niobate and vanadate species are stronger inhibitors of the SR calcium ATPase than simple monomeric vanadate, tungstate and molybdate oxometalates, thus affecting calcium homeostasis, cell signalling and cell bioenergetics, as well many other cellular processes. The ability of these oxometalates to act either as phosphate analogues, as a transition-state analogue in enzyme-catalysed phosphoryl group transfer processes and as potentially nucleotide-dependent enzymes modulators or inhibitors, suggests that different oxometalates may reveal different mechanistic preferences in these classes of enzymes.
authors.affiliation: Department of Food Engineering, ISE, University of Algarve, 8005-139 Faro, Portugal.
authors.forename: Gil
authors.initials: G
authors.lastname: Fraqueza
authors.forename: C André
authors.initials: CA
authors.lastname: Ohlin
authors.forename: William H
authors.initials: WH
authors.lastname: Casey
authors.forename: Manuel
authors.initials: M
authors.lastname: Aureliano
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006868:Hydrolysis; D008982:Molybdenum / Q000494:pharmacology*; D009556:Niobium / Q000494:pharmacology*; D010087:Oxides / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D017973:Tungsten Compounds / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology*
chemical_list: D010087:Oxides; D017973:Tungsten Compounds; C572238:decaniobate; D009556:Niobium; C044659:molybdate; D014638:Vanadates; D008982:Molybdenum; D000255:Adenosine Triphosphate; D006493:Heparin; D000252:Calcium-Transporting ATPases; C045951:tungstate