IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
The purified preparation of Spf1 P5-ATPase exhibits a Ca-stimulated ATPase activity not related to Spf1
Autor/es:
CORRADI GR, MAZZITELLI LR, PETROVICH GD, GRENON P, ADAMO HP
Lugar:
La plata
Reunión:
Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2018
Resumen:
The P5-ATPases are the most intriguing members of the large family of primary active transporters known as P-ATPases. Despite the fact that the putative transported substratehas not yet been identified, significant progress is beingmade toward the biochemical characterization of these proteins. Thebest characterized P5-ATPase is the Spf1from Saccharomyces cerevisiae.We have previously shown that purified micellar preparations of recombinant His-taggedSpf1 hydrolyzes ATP and it forms the phosphoenzymeintermediate (EP) characteristic of the transport reactioncycle of P-ATPases. Moreover,we have shown that Ca2+ modulates Spf1 by decreasingthe level of EP. Here we present results suggesting that at least part of the effect of Ca2+ is mediated by traces of contaminant proteins that co-purify with Spf1. These are as follows i) when the reaction media contained EGTA and no added Ca2+, the rate of Pi productionfrom ATP decreased with time during the first 5 min ii) the addition of Ca 2+ increased the ATPase between 0 to 5 fold depending on the preparation, and iii) Ca2+ stimulated the ATPase activity of the catalytic death mutantSpf1-D487N. The level of EP formed bySpf1 was higher inthe presence of EGTA and decreased with the increase in the Ca2+ of the media. The magnitude of this Ca2+effect on the EP level showed a positive correlationwith the stimulation of the ATPase activity in each preparation of Spf1 tested. Analysis by mass spectrometry ofthe Spf1preparation after SDS-PAGE detected the presence of several contaminant proteins. Altogether these results indicate that the activation by Ca2+of a ?contaminant?ATPasepresent inthe purified preparationof Spf1 decreases the level of Spf1 EP. Thus the effect of Ca2+ on Spf1 may result from the activation ofanother protein interacting with Spf1 and not an intrinsic feature of the enzyme.