INVESTIGADORES
CORRADI Gerardo Raul
congresos y reuniones científicas
Título:
High phosphatase activity of the recombinant Spf1 P5-ATPase
Autor/es:
NICOLAS A. CZYSEZON; GERARDO R. CORRADI; HUGO P. ADAMO
Reunión:
Congreso; XLI Reunión Anual organizada por la Sociedad Argentina de Biofísica; 2012
Resumen:
The P5-ATPases 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 P5-
ATPases are presumed to share a catalytic mechanism similar to those of
other members of the family as the Na+/K+-ATPase and the plasma
membrane Ca2+-ATPase (PMCA). However, the identity of the ion which
would be transported by the P5-ATPases remains elusive. In contrast there
are overwhelming evidences of the importance of the P5-ATPases for the
eukaryotic cell. In humans, the loss of function the ATP13A2 P5-ATPase
has been shown to cause neurodegenerative diseases like Parkinsons. 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). The purified rSpf1 is capable of
hydrolyzing ATP (2.7 μmol/mg/min) and forming a phosphoenzyme in the
virtual absence of metal ions with the exception of Mg2+ which is a known
cofactor required for all the P-ATPases. Here we have investigated the
phosphatase activity of rSpf1 by measuring the ability to hydrolyze pnitrophenylphosphate(p-NPP). At 37 °C, in the presence of 12 mM p-NPP
and 4 mM MgCl2, rSpf1 exhibited a high p-NPPase of 1.4 μmol
pNOH/mg/min. This value is similar to that of other P-ATPases when
stabilized in the E2 conformation. These results disagree with previous
measurements of the E~P formation suggesting that the enzyme was in the
E1 form (Corradi et al., 2012) and indicate that either a) a protein with
pNPPase activity is present in our preparation of purified Spf1 or b) rSpf1 hydrolyzes pNPP and ATP with similar efficiency.