IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The use of Azido Ruthenium photoactivatable probe to study the Ca2+-binding site of the Plasma Membrane Calcium Pump
Autor/es:
MALLKU ONTIVEROS; IRENE CECILIA MANGIALAVORI; MARIA FLORENCIA PIGNATARO; JORGE MARTIARENA; JUAN PABLO F.C. ROSSI; MARIELA FERREIRA -GOMES
Lugar:
Bahía Blanca
Reunión:
Congreso; XLIII. Reunión Anual de la Sociedad Argentina de Biofísica; 2014
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
The Plasma Membrane Calcium ATPase (PMCA) is a calmodulin-regulated P-type ATPase responsible for the maintenance of low intracellular concentration of Ca2+ in most eukaryotic cells. It couples the transport of Ca2+ outside the cells with the hydrolysis of ATP. Since the crystal structure of PMCA is still not resolved, the information about the Ca2+ binding site is limited to the comparative studies with the structure of Sarcoplasmic Reticulum Calcium ATPase. There are evidences that certain amino acids on the transmembrane segments M4 and M6 would be linked with the calcium binding site [1]. The purpose of this work is to identify and characterize the Ca2+-binding site of PMCA. For this, we synthesize a Ca2+-like photoactivatable reagent, azido-ruthenium (AzRu) which binds covalently and specifically to Ca2+-binding proteins after exposure to ultraviolet irradiation at 290 nm [2]. The experiments were performed with vesicles and purified PMCA obtained from human erythrocytes. Results showed that (a) AzRu inhibited Ca2+-dependent activities with high affinity; (b) AzRu had no effect on Mg2+-dependent activity and on other ATPases like the Na-K pump. The ability of AzRu to inhibit Ca2+-dependent activities was enhanced when PMCA-AzRu was exposed to irradiation, suggesting that photoactivation leads to an irreversible binding of the probe to the enzyme. These results provide a new approach to localize the Ca2+-binding site of PMCA in combination with MALDI-TOF spectroscopy. With grants of ANPCYT, CONICET and UBACYT. 1- Rinaldi D., Adamo H. Biochimica et Biophysica Acta. 2009. 2404. 2- Israelson A., Zilberberg N. & Shoshan-Barmatz, V. Nature Protocols. 2006. 111.