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

MALLKU ONTIVEROS; LUCILA GENTILE; IRENE MANGIALAVORI; JUAN PABLO ROSSI; MARIELA FERREIRA GOMES

Congreso; Latin American Conference On Mathematical Modeling Of Biological Systems.; 2015

The Plasma Membrane Calcium ATPase (PMCA) is a P-type ATPase that maintains the homeostasis of Ca2+ in eukaryotic cells. It couples the transport of Ca2+ with the hydrolysis of ATP. The crystal structure of PMCA is still not resolved, so the information is limited to data given from crystal structures of the sarcoplasmic Ca2+-pump (SERCA). In SERCA, AzRu covalently binds to the two sites for Ca2+. The purpose of this work is to identify 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+-binding proteins after exposure to irradiation at 290 nm1. The experiments were performed with inside-out vesicles2 (IOVs) and purified PMCA3 from human erythrocytes. The effect of AzRu was assayed in two different conditions: non-covalently bound (in the dark) and covalently-bound (after photolysis). Our results shown that, (a) Non-covalent AzRu inhibits completely Ca2+-ATPase activity of PMCA. When PMCA in the presence of AzRu was submitted to irradiation, 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 proteins which possess free sulfhydryl groups, prevents partially the inhibition of Ca2+-ATPase activity of PMCA, probably by reaction with Cys 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 of Mg2+ the inhibition to PMCA is lower, suggesting that the site for Mg2+ is the target of AzRu in PMCA. As a photo-activation control, AzRu was covalently bound to calmodulin. In this case we observed the formation of an adduct between AzRu and calmodulin by RP-HPLC-ESI-IT mass spectroscopy. Future experiments were designed to identify the site(s) of interaction of AzRu with PMCA by means of MALDI-TOF mass spectroscopy studies. References: [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