An Azido-Ruthenium photoactivatable probe as a strategy for the functional and structural study of the Plasma Membrane Calcium Pump

ONTIVEROS, M. Q., GENTILE, L., MANGIALAVORI, I.C, ROSSI, J.P AND FERRIRA-GOMES MS.

Salto

Congreso; Latin American crosstalk in Biophisycs and physiology; 2015

Postlatam

An Azido-Ruthenium photoactivatable probe as a strategy for the functional and structural study of the Plasma Membrane Calcium Pump. SBF.uy Ontiveros, M1; Gentile, L1; Mangialavori, I1,Rossi, J P1; Ferreira-Gomes, M11Instituto deQuímica y Fisicoquímica Biológicas. ?Prof.Paladini?. Departamento de QuímicaBiológica, Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires.mallkuontiveros@qb.ffyb.uba.ar The PlasmaMembrane Calcium ATPase (PMCA) is a P-type ATPase that maintains thehomeostasis of Ca2+ in eukaryotic cells. It couples the transport ofCa2+ with the hydrolysis of ATP. The crystal structure of PMCA isstill not resolved, so the information is limited to data given from crystalstructures of the sarcoplasmic Ca2+-pump (SERCA). In SERCA, AzRu covalentlybinds to the two sites for Ca2+. The purpose of this work is toidentify and characterize the Ca2+-binding site of PMCA. For this,we synthesized the Ca2+-like photoactivatable reagent,azido-ruthenium (AzRu) which binds covalently and specifically to Ca2+-bindingproteins after exposure to irradiation at 290 nm1. The experimentswere performed with inside-out vesicles2 (IOVs) and purified PMCA3from human erythrocytes. The effect of AzRu was assayed in two differentconditions: non-covalently bound (in the dark) and covalently-bound (after photolysis).Our results shown that, (a) Non-covalentAzRu inhibits completely Ca2+-ATPase activity of PMCA. When PMCA inthe presence of AzRu was submitted to irradiation, a higher inhibition rate wasobserved, suggesting that photo-activation leads to a covalent bond between thereagent and PMCA; (b) incubationof PMCA with AzRu in the presence of DL-Dithiothreitol; (Clelands reagent) whichis used to stabilize enzymes and other proteins which possess free sulfhydryl groups, prevents partially theinhibition of Ca2+-ATPaseactivity of PMCA, probably by reaction withCys residues of the pump; (c) when photo-activation was done in the presence of an excess of Ca2+the inhibition was similar to the control; (d) conversely, in the presence ofMg2+ theinhibition to PMCA is lower, suggesting that the site for Mg2+ isthe target of AzRu in PMCA. As a photo-activation control, AzRu was covalently bound to calmodulin. Inthis case we observed the formation of an adduct between AzRu and calmodulin byRP-HPLC-ESI-IT mass spectroscopy. Future experiments were designed to identifythe site(s) of interaction of AzRu with PMCA by means of MALDI-TOF mass spectroscopystudies.  References:  [1] IsraelsonA et al. Nature Protocols 1, 111-117, 2006 [2] Steck TL et al. Science. 168,255?257, 1970. [3] Filomatori CV and Rega AF. J Biol Chem.278, 22265-22271, 2003 Acknowledgementsand Support of ANPCYT, CONICET y UBACYT