Two different mechanisms of PMCA inhibition by flavonoids Ontiveros, M; Rinaldi, D; Pantano S; Marder, M; Mangialavori, I, Rossi, RC, Rossi, JP; Ferreira-Gomes, M Research on flavonoids from plant sources has recently sparked increasing interest because

.ROSSI, JUAN PABLO F.C.; SAFFIOTI NICOLÁS ANDRÉS; RIESCO ANA SOL; DE SAUTU MARILINA1; ROSSI ROLANDO1; BERLIN JOSHUA2; FERREIRA-GOMES MARIELA1; ROSSI JUAN PABLO1 AND MANGIALAVORI IRENE1.

SAN LUIS

Congreso; 279. XLVIII Reunión Anual SAB, 27-29 Noviembre 2019. San Luis, Argentina; 2019

SOCIEDAD ARGENTINA DE BIOFÍSICA

Conformationalselective binding of metal fluoride complexes to the Plasma Membrane Ca2+-ATPaseand structure stabilization. Saffioti Nicolás Andrés; Riesco Ana Sol; deSautu Marilina1; Rossi Rolando1; Berlin Joshua2;Ferreira-Gomes Mariela1; Rossi Juan Pablo1 and MangialavoriIrene1.  1Institutode Química y Fisicoquímica Biológicas. ?Prof.Paladini?. Departamento de QuímicaBiológica, Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires.2Departmentof Pharmacology and Physiology, New Jersey. Medical School, Rutgers University,Newark, NJ, USA The Plasma Membrane Ca2+ ATPase(PMCA) is a key participant in cytoplasmic Ca2+ regulation. Asothers P-ATPases, PMCA transports Ca2+ by the Albers and Postmechanism. In this, proteins have two main conformations, E1 and E2,and phosphorylate from ATP to transport ions against their electrochemicalgradient. Recently, we demonstrated that fluoride complexes of aluminium (AlFx),beryllium (BeFx) and magnesium (MgFx) stabilizeconformations very similar to the phosphorylated intermediate of PMCA1.The aim of this work is to understand whatconformational changes occur upon the phosphorylation of PMCA. To this end, wemeasure the fluorescence of the probe eosin, which binds with high affinity tothe nucleotide binding site of PMCA. When the protein binds fluoride complexes,eosin fluorescence decreases, and the same effect is seen when the pumpphosphorylates from ATP in presence of Ca2+. However, duringtransport activity, part of the fluorescence decrease is due to displacement ofeosin by ADP from its binding site. Our results show that the ADP and ATPaffinity for PMCA in E2 conformation are 172 ± 28 and 74 ± 17 µM, respectively.The binding of fluoride complexes to PMCA depended strictly on Mg2+concentration. Furthermore, these fluoride complexes only binds to PMCA-Mg2+.This property allowed us to measure the PMCA affinity for Mg2+ (2,2 ± 0,4 mM) in E2conformation. Finally, we studied thermal stability of PMCA by measuring theenzyme activity as a function of time. Fluoride complexes stabilize PMCAstructure and delay activity loss when the protein is incubated at 44°Ccompared to the free protein. We can conclude that fluoride complexes areligands of PMCA-Mg2+ complex, they set PMCA structure in aconformation analogous to E2-P, stabilizing it. This property allowsto study the PMCA structure in E2 conformations. 1 Saffioti N.A, de Sautu M.,Ferreira-Gomes M.S., Rossi R.C., Berlin J., Rossi J.P., Mangialavori, I.C.(2019) BBA-Biomembranes. 1861,366-379. This work was supported by Agencia Nacional dePromoción Científica y Tecnológica PICT 2014 0065, Consejo Nacional deInvestigaciones Científicas y Técnicas PIP 11220150100250CO, and Universidad deBuenos Aires Ciencia y Técnica grant 2014-2017: 20020130100254B