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

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

Córdoba

Congreso; LII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2016

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

During 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 Mg, Al and Be 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, BeF and AlF 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 BeF indicating a different enzyme conformation in these conditions. Unlike for E2P, the stability of E2BeF and E2AlF 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 BeF. Results with BeF suggest a change of the equilibria between states with bound and occluded Rb.