title: Inhibition of the Formation of the Spf1p Phosphoenzyme by Ca2.
abstract: P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeastSaccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg(2+), and EGTA. Low concentrations of Ca(2+)in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn(2+)nor Mg(2+)had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca(2+) TheKmfor ATP in the phosphorylation reaction was ∼1 μmand did not significantly change in the presence of Ca(2+) Half-maximal phosphorylation was attained at 8 μmMg(2+), but higher concentrations partially protected from Ca(2+)inhibition. In conditions similar to those used for phosphorylation, Ca(2+)had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.
authors: From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Ciudad Autónoma de Buenos Aires, Argentina.
authors: Gerardo R
authors: GR
authors: Corradi
authors: From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Ciudad Autónoma de Buenos Aires, Argentina.
authors: Nicolas A
authors: NA
authors: Czysezon
authors: From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Ciudad Autónoma de Buenos Aires, Argentina.
authors: Luciana R
authors: LR
authors: Mazzitelli
authors: From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Ciudad Autónoma de Buenos Aires, Argentina.
authors: Nicolas
authors: N
authors: Sarbia
authors: From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Ciudad Autónoma de Buenos Aires, Argentina hpadamo@qb.ffyb.uba.ar.
authors: Hugo P
authors: HP
authors: Adamo
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D010766:Phosphorylation / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; C406793:SPF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D002118:Calcium
keywords: ATPase; P-ATPase; P5-ATPase; ion pump; phosphoryl transfer; phosphorylation; protein phosphorylation; transporter