Modulation of the purified yeast P5A-ATPase Spf1p by Ca2+

HUGO P. ADAMO; GERARDO R. CORRADI; NICOLÁS A. CZYSEZON; CORRADI G R

Simposio; Na, K-ATPase and related transport ATPases: Structure, Mechanism, Cell Biology, Health and Disease; 2014

Since the earlier experiments in yeast showed that K.O. of The Sfp1gene altered Ca2+ homeostasis, Ca2+ has been a strong candidate for the ion transported by the P5A-ATPases. However, up to now the biochemistry of the SPf1p does not favor this idea. Indeed, while the ATPase activity of a typical Ca2+ transporting ATPase is stimulated more than 50 times by saturating concentrations of Ca2+, we and others (1/2) found that the response of the ATPase activity of the Spf1p was in any case much more modest than that expected for a Ca2+ pump. Recently, Ca2+ was reported to accelerate the dephosphorylation of a plant P5A-ATPase . This finding prompted us to reexamine the effects of Ca2+ on the Spf1p. The ATPase activity of the His-tag purified Spf1p was measured at 37º C in a reaction medium containing 50Mm Tris-CIH Ph 7.7 at 4 ºC, 3 Mm (32p)-ATP, 2 Mm Mg2+, 80 Mm KCI, 0,5 Mm EGTA, 0,034 % C12E10, 0,057 % PC, 90 mM imidazole and 1.5 ug of GPS-Spf1p, with or without enough Ca2CI to give 10 Um Ca2+ free. In the EGTA medium the Pi liberated increased rapidly with time up to 2.5 min and then remained nearly constant. In contrast, in the presence of Ca2+ the Pi increased slowly up to 15 min. The initial velocities calculated at 0.25 min were 8 and 2.5 umol/mg/min in the EGTA and in the Ca2+ medium respectively. The formation of EP was maximal in EGTA and decreased in the presence of the uM concentrations of Ca2+. These results suggest the existence of a regulatory mechanism by which Ca2+, at concentrations in the physiological range, decrease the activity of the Spf1p ATPase.