Interaction between PMCA and unsaturated fatty acids: The use of a photoactivatable oleic acid analog.

MARIA FLORENCIA PIGNATARO; IRENE MANGIALAVORI; JOSÉ MARÍA DELFINO; JUAN PABLO ROSSI

San Miguel-Tucuman

Congreso; XLI. Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2012

Sociedad de Biofísica Argentina

The lipid environment plays a central role in the regulation of integral membrane proteins biological function. The modulation provided by the surrounding phospholipids, both neutral or charged, has been extensively described whereas some knowledge regarding regulation by unsaturated fatty acids (UFAs) still remains to be gained.  As an integral membrane protein example to study this regulation, we chose plasma membrane Ca2+-ATPase (PMCA) which is responsible for maintaining the low calcium levels in resting cells. Niggli et al1 presented one of the first evidences regarding the UFAs regulation over PMCA: Oleic acid increases both Vmax and Ca2+ affinity. The purpose of this work was to further characterize the mechanisms involved in the modulation exerted by UFAs on PMCA. With this aim, we studied the interaction between purified PMCA of human red blood cells and a photoactivatable oleic acid analog, 8-(5´-azido-O-hexanoyl-salicylamido)octanoic acid (AS86)2. Our results showed that AS86 interacts non-covalently with PMCA showing a biphasic behaviour, similar to that observed for the effect of oleic acid1. Whenever AS86 interacts covalently with PMCA, it has no effect on the ATPase activity in the presence of Ca2+ alone, however, when Calmodulin (CaM) is present, increasing concentrations of this probe, generates an inhibitory effect on PMCA activation by CaM. In order to provide some insights in the elucidation of the region where UFAs may interact with this enzyme, we performed several photolabeling experiments with this probe, to consequently subject the sample to a mass spectrometer analysis (MALDI-TOF), finding differential labeling in distinct domains of PMCA.     1. Referencias: 1Niggli,V.,Adunyah, E.S., & Carafoli, E. (1981) J. Biol. Chem. 256, 8588-8592 2Rossi, J.P.,Delfino, J.M., Caride, A.J., & Fernández ,H.N. (1995) Biochemisty,31-11:3802-12 Con subsidios de ANPCyT, CONICET, UBA