| Project/Area Number |
14580639
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Asahikawa Medical College |
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Principal Investigator |
SUZUKI Hiroshi Asahikawa Medical College, Biochemistry, professor, 医学部, 教授 (50183421)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Sanae Asahikawa Medical College, Biochemistry, Assistant, 医学部, 助手 (80291061)
YAMASAKI Kazuo Asahikawa Medical College, Biochemistry, Assistant, 医学部, 助手 (60241428)
DAIHO Takashi Asahikawa Medical College, Biochemistry, Assistant professor, 医学部, 助教授 (90207267)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Endplasmic reticulum / Ca^<2+>-ATPase / Ca^<2+> pump / Darier's disease / SERCA / P-type ATPase / Active transport / Phosphorylate intermediate / Ca ^<2+>ポンプ / Ca ^<2+>-ATPase / ドメイン構造 / ドメイン相互作用 / 遺伝病 / Ca ^<2+>動態 |
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| Research Abstract |
Sarcoplasmic reticulum Ca^<2+>-ATPase (SERCA1a) catalyzes Ca^<2+> transport coupled with ATP hydrolysis. We previously showed that the large rotation of A domain and its intimate contact with P and N domains most likely occur during the isomeric transition of phosphorylated intermediate(E1PCa_2 to E2PCa_2 transition) and Ca^<2+> release to form the most compactly organized single headpiece in the Ca^<2+>-released form of E2P, and suggested that stabilization energy provided by the intimate contacts between 3 cytoplasmic domains in E2P will provide energy for moving transmembrane helices and release the bound Ca^<2+> into lumen. In this research, we further explored the regions and residues responsible for the movements of and interactions between the domains by site-directed mutagenesis and kinetic analysis with the mutants, and found follows.
1.The Glu^<40>-Ser^<48> loop with its proper length(connecting A domain and 1st transmembrane helix(M1)) is critical for the large rotation of A domain and coordinated motion of M1 during the E1PCa_2 to E2PCa_2 transition.
2.Arg334 on the top of M4(connected to P domain)and Tyr122 on the top of M2(connected to A domain)play essential roles in the E1PCa_2 to E2PCa_2 transition and in the subsequent Ca^<2+> release form and hydrolysis of E2P, respectively, by contributing to the motions of and interactions between A and P domains.
3.The outermost Val200 loop of A domain is one of the regions responsible for the intimate contact between A and P domains in E2P to release bound Ca^<2+> into lumen.
We also developed the stable E2P analogue and conditions to solubilize, purify, and stabilize the analogue for the crystallization. We also revealed the effects of mutations on A domain responsible for Daner's disease, Δ41,Δ42,N39D, and N39T on the kinetic properties in order to understand the molecular mechanism of the disease.
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