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

ONTIVEROS, MALLKU; GENTILE, LUCILA; MANGIALAVORI IRENE; ROSSI, RC; FERREIRA GOMES, MARIELA

Salto

Congreso; Latin American Crosstalk In Biophysics And Physiology; 2015

The Plasma Membrane Calcium ATPase (PMCA) is a P-type ATPase thatmaintains the homeostasis of Ca2+ in eukaryotic cells. It couples thetransport of Ca2+ with the hydrolysis of ATP. The crystal structure of PMCAis still not resolved, so the information is limited to data given from crystalstructures of the sarcoplasmic Ca2+-pump (SERCA). In SERCA, AzRucovalently binds to the two sites for Ca2+. The purpose of this work is toidentify and characterize the Ca2+-binding site of PMCA. For this, wesynthesized the Ca2+-like photoactivatable reagent, azido-ruthenium (AzRu)which binds covalently and specifically to Ca2+-binding proteins afterexposure to irradiation at 290 nm1inside-out vesicles2The effect of AzRu was assayed in two different conditions: non-covalentlybound (in the dark) and covalently-bound (after photolysis). Our resultsshown that, (a) Non-covalent AzRu inhibits completely Ca2+-ATPaseactivity of PMCA. When PMCA in the presence of AzRu was submitted toirradiation, a higher inhibition rate was observed, suggesting that photo-activation leads to a covalent bond between the reagent and PMCA; (b)incubation of PMCA with AzRu in the presence of DL-Dithiothreitol;(Clelands reagent) which is used to stabilize enzymes and other proteinswhich possess free sulfhydryl groups, prevents partially the inhibition ofCa2+-ATPase activity of PMCA, probably by reaction with Cys residues ofthe pump; (c) when photo-activation was done in the presence of an excessof Ca2+ the inhibition was similar to the control; (d) conversely, in thepresence of Mg2+ the inhibition to PMCA is lower, suggesting that the sitefor Mg2+ is the target of AzRu in PMCA. As a photo-activation control,AzRu was covalently bound to calmodulin. In this case we observed theformation of an adduct between AzRu and calmodulin by RP-HPLC-ESI-ITmass spectroscopy. Future experiments were designed to identify the site(s)of interaction of AzRu with PMCA by means of MALDI-TOF massspectroscopy studies.[1] Israelson A 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, 2003Acknowledgements and Support of ANPCYT, CONICET y UBACYT. The experiments were performed with(IOVs) and purified PMCA3from human erythrocytes.