| Project/Area Number |
15590721
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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 |
Circulatory organs internal medicine
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YAMASHITA Hiroshi The University of Tokyo, Faculty of Medicine, Assistant, 医学部附属病院, 助手 (50323572)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIURA Seiryo The University of Tokyo, Graduate School of Frontier Sciences, Professor, 大学院・新領域創成科学研究科, 教授 (10272551)
SATA Masataka The University of Tokyo, Faculty of Medicine, Assistant professor, 医学部附属病院, 助教授 (80345214)
KATOH Masayoshi The University of Tokyo, Faculty of Medicine, Medical Staff, 医学部附属病院, 医員
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Cardiac myosin / Myosin light chain kinase / Myosin light chain phosphorylation / Laser trap / In vitro motility assay / Actin-activated ATPase activity / Telokin / Subfragment-2 / ミオシン軽鎖アイソフォーム / カーボンファイバー / ミオシン連鎖リン酸化 / 細胞内カルシウム濃度 / 単離心筋細胞 |
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| Research Abstract |
Myosin light chain kinase (MLCK) is a multifunctional protein with kinase domain in the middle, actin-binding domain in the amino-terminus, and myosin binding domain in the carboxyl-terminus of the molecule. In smooth muscles, MLCK phosphorylates the regulatory light chain (RLC) to activate myosin ATPase activity and start muscle shortening. In cardiac muscles, however, MLCK also phosphorylates RLC and has a modulatory effect on muscle contraction, i.e. phorphorylation of RLC increases calcium sensitivity of isometric force production. Recently, Kohama et al. reported that MLCK could enhance molecular function by directly binding to smooth muscle myosin molecules without phosphorylating RLC. Although similar MLCK isoform is expressed and localized on the myosin filaments in cardiac muscles, the functional role of MLCK is not well elucidated. Therefore, we investigated the effect of MLCK on the molecular function of cardiac myosin in vitro.
An amino-terminal MLCK fragment (MF) containing
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myosin-binding region but lacking the kinase domain was expressed in E.coli using bovine stomach MLCK cDNA and purified. Myosin was purified from rat cardiac muscles. Actin-activated ATPase activity and actin filament velocity in the in vitro motility assay were measured in the presence and absence of 5mmol/L of MF. The filament velocity was 22% higher in the presence of MF compared to the control condition (5.6±0.5 v.s. 4.6±0.4 mm/sec, p<0.01). The ATPase activity was not different between these conditions. On the urea polyacrylamide gels, the phosphorylation level was not affected by the addition of MF, either.
The MLCK fragment studied in the present study is expressed in smooth muscle cells as an independent protein, telokin. In smooth muscles, telokin is proposed to bind to the S-1-S-2 junction of myosin molecule and induce a conformational change. Taken together, the present results suggest that MLCK may modulate cardiac function not only by phosphorylating RLC to increase calcium sensitivity but also by directly binding to the myosin molecule to change conformation. Less
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