Details

Autor(en) / Beteiligte

• Montigny, Cedric
• Jaxel, Christine
• Shainskaya, Alla
• Vinh, Joëlle
• Labas, Valérie
• Møller, Jesper V
• Karlish, Steven J D
• le Maire, Marc

Titel

Fe2+-catalyzed Oxidative Cleavages of Ca2+-ATPase Reveal Novel Features of Its Pumping Mechanism

Ist Teil von

• The Journal of biological chemistry, 2004-10, Vol.279 (42), p.43971-43981

Ort / Verlag

United States: American Society for Biochemistry and Molecular Biology

Erscheinungsjahr

2004

Quelle

MEDLINE

Beschreibungen/Notizen

• We have analyzed the Fe 2+ -catalyzed oxidative cleavages of Ca 2+ -ATPase in the presence of Ca 2+ , with or without the ATP analog 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na + ,K + -ATPase or other P-type pumps and are expected on the basis of the putative Mg 2+ binding residues near the phosphorylated Asp 351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe 2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na + ,K + -ATPase and H + ,K + -ATPase. This site could represent a Ca 2+ binding zone between M1 and M3, preceding Ca 2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca 2+ (E1Ca 2 conformation), whereas the presence of Mg·ATP stabilizes the N to P interaction (E1·Mg·ATP conformation).