The purified preparation of Spf1 P5-ATPase exhibits a Ca-stimulated ATPase activity not related to Spf1

CORRADI GERARDO RAUL; MAZZITELLI LUCIANA ROMINA; PETROVICH GUIDO DANIEL; GRENON PAULA; ADAMO HUGO PEDRO

La Plata, Argentina

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

The  P5-ATPases are the most intriguing members of  the  large  family of  primaryactive transporters known asP-ATPases. Despite the fact that the putativetransported 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 itforms  the  phosphoenzymeintermediate  (EP)characteristic of the transport reactioncycle  of  P-ATPases. Moreover,we  haveshown that Ca2+ modulates Spf1  by  decreasingthe  level  of  EP. Here we present results suggesting that  at least part ofthe  effect of Ca2+ is mediated by traces  ofcontaminant 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 productionfromATPdecreased 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 activityof the catalytic death mutantSpf1-D487N. The level of EP formed bySpf1 was higher inthe  presence of EGTA and decreased with  theincrease 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 activityin  each preparationof Spf1 tested.Analysis by mass spectrometryofthe  Spf1preparation after SDS-PAGE detectedthe  presence of severalcontaminant 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 theeffect of Ca2+ on Spf1 may  result from the activation ofanother protein interactingwith  Spf1 and not an intrinsic feature of the enzyme.