CONICET | Buscador de Institutos y Recursos Humanos

The action of natural flavonoids as modulators of the catalytic activity of enzymes has been attributed to various related chemical species, such as the formation of complexes with metals or even oxidized forms thereof. We recently demonstrated that the flavonoids quercetin and gossypin inhibit the Ca2+-ATPase activity of plasma membrane calcium ATPase (PMCA) as a function of Mg2+ concentration, suggesting that the true inhibitor is quercetin-Mg2+ or gossypin-Mg2+. The aim of this work was to investigate the formation and stability of the quercetin-Mg2+ and gossypin-Mg2+ complexes and their effects on the inhibition of the activity of PMCA. We investigated the formation and stability of flavonoid-Mg2+ complexes using UV-Vis spectroscopy. The results show that flavonoids and their Mg2+ complexes in MOPs buffers (pH 7.4) were unstable and oxidized species were produced. These results are in agreement with the studies carried out by mass spectrometry. On the other hand, the formation and stability of the flavonoid-Mg2+ were examined in the same medium in which the PMCA activity was carried out. This medium contained the reagents necessary for the determination of activity, including mixed micelles of C12E10 and DMPC. The results show that quercetin, gossypin, and their Mg2+ complexes remained stable without the appearance of oxidized species, suggesting that the presence of lipids increases the stability of flavonoids. Our results show that quercetin-Mg2+ and gossypin-Mg2+ complexes were formed with a stoichiometry of 1:1 and a Kd of 13.5 ± 2.7 mM and 19.6 ± 1.4 mM, respectively. On this basis, the Mg2+-dependent inhibition of PMCA activity was expressed as a function of the concentrations of quercetin-Mg2+ and gossypin-Mg 2+ showing that PMCA inhibition depends on the concentration of the flavonoid-Mg2+ complex, suggesting that these are the true PMCA inhibitors.

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