Is the ATPase activity of the P5-ATPase rSpf1 stimulated by Ca2+?

CZYSEZON, N. A; CORRADI, G. R; ADAMO, H. P.A.

cordoba

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

The transporters known as P5-ATPases have been associated with the early development of Parkinson´s disease and other neurodegenerative disorders. They belong to the family of ATP fuelled ion pumps which cycle between two major conformations E1 and E2 and form an acylphosphate (E~P) species as intermediate during transport. The ion transported by the P5-ATPases is not known. We have recently begun to characterize the catalytic cycle of the P5-ATPase Spf1 from Saccharomyces cerevisiae obtained by overexpression in the same yeast (Corradi et al., 2012). Here we have investigated the effect of Ca2+ on the ATPase activity of the pump, as well as its role in the EP formation. At 37° C and in the presence of 3 μM Ca2+ the purified recombiant Spf1 with the GFP fused to its N-terminus (rSpf1) was capable of hydrolyzing ATP at a nearly linear rate of about 5.7 μmol Pi/mg min for 7.5 minutes. In contras in the absence of Ca2+ (0.5 mM EGTA) the ATPase activity rapidly decreased. Maximum ATPase activty was achieved in the presence of 3 μM Ca2+ and remained high at concentrations of Ca2+ up to 1 mM. The incubation of rSpf1 with ATP for 30 s at 4° C resulted in the formation of the phosphoenzyme intermediate. Noteworthy, the formation of the EP intermediate was higher in the presence of EGTA and decreased in the presence of 10 μM Ca2+. If the ATPase activity and EP formation are both ascribed to the rSpf1 enzyme, these results may indicate that Ca2+ stimulates the ATPase activity of rSpf1 by accelerating the turnover of EP. Acknowledgments: This work is being supported by ANPCyT Prestamo BID 2134, UBACYT y CONICET. CONICET PIP 2022.