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.