CONICET | Buscador de Institutos y Recursos Humanos

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 structure of PMCA is still not solved, and only limited information is available of ligand binding sites. The purpose of this work is to identify and characterize the calcium binding site of PMCA. We synthesized azido-ruthenium (AzRu), a photoactivatable reagent designed to obtain structural information, which binds covalently and specifically to Ca2+-binding proteins after irradiation at 290 nm. The experiments were performed with purified PMCA from human erythrocytes. The results show that AzRu irreversibly inhibits PMCA activity. The time course of production of inorganic phosphate was measured before and after the addition of AzRu, in conditions where photolysis of the inhibitor was prevented. It was observed that the rate of inhibition by AzRu decreases by increasing [Ca2+] in the medium, whereas this rate remains unaffected by increasing [Mg2+]. These results suggest that the calcium binding site would be involved in the inhibition process. Assays with [γ-32P] ATP indicate that inhibition of the ATPase activity was accompanied by an increase in the phosphorylated intermediate levels, which suggests that AzRu could be blocking the dephosphorylation of the pump. The photolabeling experiments with AzRu were observed by mass spectrometry using calmodulin (CaM) and lysozyme. CaM-AzRu adducts were observed but not with lysozyme. The results confirm the specific interaction of AzRu with calcium dependent proteins. Photolabeling mass spectra of PMCA with AzRu showed the disappearance of signals from certain peptides from the cytoplasmic domains suggesting an interaction with AzRu. Due to the difficulty of studying membrane proteins by mass spectrometry, we are evaluating an optimization of the study of the transmembrane domain where the PMCA-AzRu adduct would be found.