Interaction of an ATP analogue with the Plasma Membrane Calcium Pump

NICOLÁS SAFFIOTI; MARIELA FERREIRA-GOMES; JUAN PABLO F.C. ROSSI; IRENE MANGIALAVORI

Congreso; XLII. Reunión Anual de la Sociedad Argentina de Biofísica; 2013

The Plasma Membrane Calcium Pump (PMCA) is a highly regulated protein present in all eukaryotic cells. It belongs to the P-type family, a group that shares the characteristic of ion transport coupled to the ATP hydrolysis involving phosphorylated intermediates during its reaction cycle. In order to study the conformational changes in the nucleotide binding domain, we assay the 2',3'-O-(2,4,6-Trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) an ATP analogue with fluorescence properties with purified PMCA. We have previously shown that TNP-ATP binds to the protein increasing its quantum yield allowing the detection of conformational changes among intermediates of the reaction cycle1. In this work we characterize the interaction between TNP-ATP and PMCA purified from human red cells in equilibrium and in steady-state conditions. To this purpose, Ca2+-ATPase activity and TNP-ATP fluorescent emission were measured in the presence or absence of calmodulina or phosphatidylserine. Our results show that PMCA do not hydrolyze TNP-ATP. This ATP analogue behaves as a pure competitive inhibitor of the enzyme. Its apparent Ki is not significantly modified when the pump is incubated in the presence of activators. Results show that (1) TNP-ATP binds to the same site of ATP and (2) the increase of PMCA activity by calmodulin or phosphatidylserine do not modify the binding site of the nucleotide.   With grants of ANPCYT, CONICET, UBACYT y NIH   1- Mangialavori IC, Ferreira-Gomes MS, Saffioti NA, Gonzalez-Lebrero RM, Rossi RC, Rossi JP. J Biol Chem 2013. 2013. In the Press