| Autor: |
Jesper V, Möller, Guillaume, Lenoir, Christophe, Marchand, Cédric, Montigny, Marc, le Maire, Chikashi, Toyoshima, Birte Staehr, Juul, Philippe, Champeil |
| Rok vydání: |
2002 |
| Předmět: |
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| Zdroj: |
The Journal of biological chemistry. 277(41) |
| ISSN: |
0021-9258 |
| Popis: |
After treatment of sarcoplasmic reticulum Ca(2+)-ATPase with proteinase K (PK) in the presence of Ca(2+) and a protecting non-phosphorylated ligand (e.g. adenosine 5'-(beta,gamma-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE(243) sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca(2+) and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADP-sensitive phosphoenzyme, Ca(2)E1P, with occluded Ca(2+). The ability of Ca(2)E1P to become converted to the Ca(2+)-free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca(2+). PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca(2+). It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca(2+) ions. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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