Kinetic characterization of a possible Ca2+ transport intermediate in the plasma membrane Ca2+-ATPase.

MARIELA, FERREIRA GOMES; MC DE LA FUENTE; RODOLFO MARTÍN GONZÁLEZ-LEBRERO; RC ROSSI; JPFC ROSSI

Montevideo

Congreso; 6th International Conference on Biological Physics and the 5th Southern Cone Biophysics Congress; 2007

The Plasma Membrane Calcium ATPase (PMCA) is a calmodulin-regulated P-type ATPase responsible for the maintenance of low intracellular concentrations of Ca2+ in most eukaryotic cells. It couples the transport of Ca2+ out of the cells with the hydrolysis of ATP into ADP and inorganic phosphate. The current kinetic model proposes that the enzyme exists in two main conformations, E1 and E2. After binding of intracellular Ca2+ to high-affinity sites, E1 can be phosphorylated by ATP with formation of the intermediate E1P, which would result in occlusion of bound Ca2+. After a conformational transition to E2P, Ca2+ would be released to the extracellular medium from low-affinity sites, the phosphoenzyme is hydrolyzed and the resulting E2 intermediate state undergoes a new conformational transition to E1. We have developed a procedure to measure Ca2+-occlusion in microsomal preparations of PMCA. PMCA was produced by Baculovirus–mediated expression in Sf9 insect cells and isolated as microsomal membranes. As we reported earlier (de la Fuente et al., 2005), we have detected occlusion of Ca2+ ions in this microsomal preparation of PMCA using the method of Rossi et al. which combines a quench-flow apparatus with a rapid-filtration device with a time resolution of 3.5 ms. Tight binding of Ca2+ was measured as a function of time in media containing different amounts of a microsomal preparation of PMCA (50-100 mg/ml), 100 mM (45Ca)Ca2+, in the presence or absence of 3 mM Mg2+, and with or without 50 mM LaIII. LaIII decrease the speed of conformational change E1P → E2P, producing an accumulation of E1P, state in which Ca2+ would occlude. The experiments were performed with or without (control) 2 mM ATP. Results show that the binding of Ca2+ can be described by the sum of two increasing exponential functions of time with of approximately the same size and apparent rate constants of about 0,1 and 0,006 sec-1. The levels of Ca2+ binding in the presence of 3 mM Mg2+ are five fold greater than in the absence of this ion. Thermal inactivation of the microsomal preparation of PMCA decreased the Ca2+ binding to a minimum.