Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca2+-dependent phosphatase
| Autor: | Paula Grenon, Michael G. Palmgren, Luciana R. Mazzitelli, Felicitas de Tezanos Pinto, Guido Daniel Petrovich, Hugo P. Adamo, Danny Mollerup Sørensen, Gerardo Raul Corradi |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Adenosine Triphosphatase ATPase Mutant Cell Membranes Yeast and Fungal Models Naphthols Biochemistry Nitrophenols purl.org/becyt/ford/1 [https] chemistry.chemical_compound Calcium Chloride 0302 clinical medicine Adenosine Triphosphate PHOSPHOENZYME Contaminants Phosphorylation Post-Translational Modification Materials Multidisciplinary biology Chemistry Triazines Organic Compounds Hydrolysis Eukaryota P5-ATPASE Ligand (biochemistry) Recombinant Proteins Enzymes Experimental Organism Systems Physical Sciences Saccharomyces Cerevisiae Medicine Cellular Structures and Organelles Research Article Saccharomyces cerevisiae Proteins Science Phosphatase Saccharomyces cerevisiae Materials Science Spf1p Research and Analysis Methods 03 medical and health sciences Saccharomyces Organophosphorus Compounds Model Organisms Ca2+ DEPENDENT PHOSPHATASE purl.org/becyt/ford/1.6 [https] Imidazole Enzyme Assays Organic Chemistry Phosphatases Organisms Fungi Chemical Compounds Substrate (chemistry) Biology and Life Sciences Proteins Membrane Proteins Cell Biology biology.organism_classification Yeast EGTA 030104 developmental biology Mutation biology.protein Biocatalysis Enzymology Animal Studies ATP-Binding Cassette Transporters 030217 neurology & neurosurgery |
| Zdroj: | PLoS ONE, Vol 15, Iss 4, p e0232476 (2020) Corradi, G R, Mazzitelli, L R, Petrovich, G D, Grenon, P, Sørensen, D M, Palmgren, M, De Tezanos Pinto, F & Adamo, H P 2020, ' Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca 2+-dependent phosphatase ', PLoS ONE, vol. 15, no. 4, e0232476 . https://doi.org/10.1371/journal.pone.0232476 CONICET Digital (CONICET) Consejo Nacional de Investigaciones Científicas y Técnicas instacron:CONICET PLoS ONE |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0232476 |
| Popis: | P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase. Fil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Mazzitelli, Luciana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Petrovich, Guido Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Grenon, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Sørensen, Danny M.. Universidad de Copenhagen; Dinamarca Fil: Palmgren, Michael. Universidad de Copenhagen; Dinamarca Fil: de Tezanos Pinto, Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina |
| Databáze: | OpenAIRE |
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