Metal fluoride complex of plasma membrane calcium pump: Effect of fluoride-stabilized phosphoenzyme analogues

SICILIANO, N., FERREIRA-GOMES, M., ROSSI, JPFC. AND MANGIALAVORI, IC.

Villa Carlos Paz, Córdoba

Congreso; ? XLII. Reunión Anual de la Sociedad Argentina de Biofísica (SAB).; 2013

SAB

Metal fluoride complex of plasma membrane calcium pump: Effect of Fluoride-stabilized phosphoenzyme analogues Siciliano, N., Ferreira-Gomes, M., Rossi, JP. and Mangialavori, IC. IQUIFIB-Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, UBA   The plasma membrane calcium pump (PMCA) belongs to the P-type ATPase family of active cation pumps. According to the conventional E1?E2 theory, E1 and E2 respectively refer to high-affinity and low-affinity states to Ca2+. Gating of the ion pathway is coupled to the phosphorylation and dephosphorylation of the ATPase. Phosphoryl transfer from ATP to an Asp in the cytoplasmic domain (i.e., E1 to E1P) closes the cytoplasmic gate, and the release of ADP triggers a change in affinity of the Ca2+ binding sites (i.e., E1P to E2P) and opening of the luminal gate. Hydrolysis of the aspartylphosphate (E2P to E2) closes the gate. Metal fluorides like magnesium-, beryllium-, and aluminum fluorides act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Thus, MgFx, AlFx, and BeFx stabilize analogues of the E2P product state (E2MgF42-), the E2P transition state (E2AlF4-), and the E2P ground state (E2BeF3-), respectively. The interaction of PMCA with these complexes has never been characterized; therefore in the present work we study the interactions of various metal fluorides (MeF) with purified preparations of PMCA. Our results show that (1) PMCA Ca2+-ATPase activity is totally inhibited in the presence of MeF (2) Inhibition of PMCA Ca2+-ATPase activity in the presence of NaF and as a function of AlCl3or BeCl2 concentrations, was well described by a rectangular hyperbola while as a function of MgCl2 a more complex behavior was observed. (3) The apparent inhibitory affinity of PMCA for these MeF was AlF4- < BeF3- <MgF42- and, (4) inhibition was highly dependent of the pH of the reaction medium.   With grants of ANPCYT, CONICET, UBACYT y NIH