Beryllium and aluminium fluoride complexes to study phosphorylated states in the Na,K-ATPase

MALÉN SAINT MARTIN; MERCEDES CENTENO; ROLANDO CARLOS ROSSI; MÓNICA MONTES

Córdoba

Congreso; Reunión Anual Sociedad Argentina de Bioquímica y Biología Molecular; 2016

SAIB

Beryllium and aluminum fluoride complexes to study phosphorylated states in the Na,K-ATPase Malén Saint Martin, Mercedes Centeno, Rolando Rossi, Mónica MontesDuring the transport of Na+ and K+ driven by the hydrolysis of ATP, Na,K-ATPase undergoes reactions of phosphorylation-dephosphorylation and conformational changes that allow the transient occlusion of the transported cations. Fluoride complexes with metals such as Mg2+, Al3+ and Be2+ have been employed for structural analysis since they bind to the phosphorylation site and imitate different sub-states of the phosphoryl intermediate, E2P. Using enzyme partially purified and the fluorescent probes eosin and RH421, which respectively report on E1 and E2P-like states, we performed equilibrium and transient-state experiments. We observed a decrease in eosin fluorescence signal due to Pi, BeFx and AlFx addition, suggesting the formation of E2P or E2P-like states. Fluorescence changes analyzed with the RH421 probe are parallel to those observed with eosin for the case of Pi but not for BeFx indicating a different enzyme conformation in these conditions. Unlike for E2P, the stability of E2BeFx and E2AlFx is sufficient to prevent the enzyme to return to the E1 state upon Na+ addition. Regarding the ability of E2P and E2P-like states to bind and occlude cations, it is observed that K+ (Rb+) occlusion is decreased by both Pi and BeFx. Results with BeFx suggest a change of the equilibria between states with bound and occluded Rb+.With grants from CONICET, ANPCYT y UBACYT.