1989 Fiscal Year Annual Research Report

分子標識・重原子標識を用いたクライオ電子顕微鏡法による筋収縮機構の研究

Research Project

Project/Area Number	61065001
Research Institution	University of Tokyo
Principal Investigator	若林健之東京大学, 理学部, 教授 (90011717)
Co-Investigator(Kenkyū-buntansha)	須藤和夫東京大学, 教養学部, 助教授 (20111453)
Keywords	アクチン / ミオシン / 電子顕微鏡法 / 筋肉収縮 / 抗体 / ビオチン / クライオ電子顕微鏡 / 重原子標識
Research Abstract	筋肉収縮・原形質流動・細胞分裂などは分子レベルではすべて、アクチンとミオシンの相互作用に依存している。この重要な相互作用の様式については国際的論争があったが、我々の解釈が国際的に受け入れられ、生体の運動の分子メカニズムの基本を担うアクチン/ミオシンの結合部位を日本人の手で決定できた。新しい方法としてアビジン・ビオチンを用いた、ATPなどを電子顕微鏡で直視出来る分子標識法を開発した。さらに金原子クラスターを用いた重原子標識クライオ電子顕微鏡法により、無染色・無固定でタンパク質構造を明らかにしつつ、標識されたアミノ酸残基の立体的位置を決定する重原子標識クライオ電子顕微鏡法を開発し、アクチンのCys374の三次元的位置を決定した。また部位特異的抗体によるミオシン頭部のマッピングにも成功した。更に、極低電子線量電子顕微鏡法の開発に成功し、イメージング・プレートを用いて電子線量をフィルム法の五分の一以下にまで引き下げ、且つ、分解能を保つことが出来る。また、100μMのADPと1mM無機リン酸の存在下ではフィラメントの滑り速度が約十分の一になり、きわめて少量のATP(1 nM)で、しかも長時間続くことを見いだした。光分解性ATPを用いた時間分解能をもつ電子顕微鏡法は、現在セットアップが完成して、いよいよデータが出始める段階に入っている。また変異アクチン遺伝子の発現やHMM(heavy meromyosin)遺伝子の発現に成功しているので、今後この部位特定的重原子標識電子顕微鏡法を完成するための要素技術はすべて揃った。これら全てを組み合わせると、アクチン/ミオシンの特定の残基を重原子標識し、それらが筋収縮の際にどのように動くかを追跡する事ができ、筋収縮の分子メカニズムをアミノ酸残基のレベルで明らかに出来るであろう。

Research Products
(17 results)

All Other

All Publications (17 results)

[Publications] K.Sutoh: "Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog." Proc.Natl.Acad.Sci.USA. 83. 212-216 (1986)
[Publications] K.Sutoh: "Actin-fragmin interactions as revealed by chemical crosslinking." Biochemistry. 25. 435-440 (1986)
[Publications] K.Sutoh: "Improved method for mapping the binding site of an actin binding protein in the actin sequence." Biochemistry. 25. 6186-6192 (1986)
[Publications] K.Sutoh: "Identification of two segments of myosin subfragment 1 heavy chain that can be crosslinked to the SH1 thiol." Biochemistry. 26. 4511-4516 (1987)
[Publications] K.Sutoh: "A short hydrophobic segment next to Trp130 in myosin heavy chain is close to the ribose ring of ADP bound in the ATPase site." Biochemistry. 26. 7648-7654 (1987)
[Publications] M.Suzuki: "Monoclonal Antibody which Recognizes Trimethylated N-Terminus of Histone H2B." Proc.Japan Acad.63. 385-388 (1987)
[Publications] M.Tokunaga: "Position of the Amino-terminus of Myosin Light Chain-1 and Light Chain-2 determined by Electron Microscopy Using Monoclonal Antibody." J.Mol.Biol.194. 245-255 (1987)
[Publications] K.Sutoh: "Electron Microscopic Visualization of the N-Terminus of the Myosin Heavy Chain using a Site-directed Antibody." J.Mol.Biol.195. 953-956 (1987)
[Publications] M.Tokunaga: "Location of the ATPase site of myosin determined by three-dimensional electron microscopy." Nature. 329. 635-638 (1987)
[Publications] K.Sutoh: "Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1." Biochemistry. 27. 2964-2969 (1988)
[Publications] M.Suzuki: "Packing of DNA in Cricket Sperm.A Compact Mode of DNA Packaging." J.Mol.Biol.204. 653-661 (1988)
[Publications] K.Hirose: "Thin filaments of Rabbit Skeletal Muscle are in Helical Register." J.Mol.Biol.204. 797-801 (1988)
[Publications] K.Sutoh: "End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin." Biochemistry. 28. 102-106 (1989)
[Publications] K.Sutoh: "End-labeling fingerprinting shows that the N- and C-termini of actin are in the contact site with gelsolin." Biochemistry. 28. 5269-5275 (1989)
[Publications] K.Sutoh: "Electron Microscopic Mapping of Myosin Head with Site-directed Antibodies." J.Mol.Biol.206. 357-363 (1989)
[Publications] K.Saeki: "New Method to Prepare single-Headed Heavy Meromyosin with High Purity and a High Yield." J.Biochem.106. 606-611 (1989)
[Publications] T.Wakabayashi: "Three-Dimensional Image Reconstruction from Electron Micrographs of Biological Macromolocules." J.Electron Microsc.38. S102-S104 (1989)

1989 Fiscal Year Annual Research Report

分子標識・重原子標識を用いたクライオ電子顕微鏡法による筋収縮機構の研究

Principal Investigator

若林 健之 東京大学, 理学部, 教授 (90011717)

Research Products

[Publications] K.Sutoh: "Electron microscopic visualization of the ATPase site of myosin by photoaffinity labeling with a biotinylated photoreactive ADP analog." Proc.Natl.Acad.Sci.USA. 83. 212-216 (1986)

[Publications] K.Sutoh: "Actin-fragmin interactions as revealed by chemical crosslinking." Biochemistry. 25. 435-440 (1986)

[Publications] K.Sutoh: "Improved method for mapping the binding site of an actin binding protein in the actin sequence." Biochemistry. 25. 6186-6192 (1986)

[Publications] K.Sutoh: "Identification of two segments of myosin subfragment 1 heavy chain that can be crosslinked to the SH1 thiol." Biochemistry. 26. 4511-4516 (1987)

[Publications] K.Sutoh: "A short hydrophobic segment next to Trp130 in myosin heavy chain is close to the ribose ring of ADP bound in the ATPase site." Biochemistry. 26. 7648-7654 (1987)

[Publications] M.Suzuki: "Monoclonal Antibody which Recognizes Trimethylated N-Terminus of Histone H2B." Proc.Japan Acad.63. 385-388 (1987)

[Publications] M.Tokunaga: "Position of the Amino-terminus of Myosin Light Chain-1 and Light Chain-2 determined by Electron Microscopy Using Monoclonal Antibody." J.Mol.Biol.194. 245-255 (1987)

[Publications] K.Sutoh: "Electron Microscopic Visualization of the N-Terminus of the Myosin Heavy Chain using a Site-directed Antibody." J.Mol.Biol.195. 953-956 (1987)

[Publications] M.Tokunaga: "Location of the ATPase site of myosin determined by three-dimensional electron microscopy." Nature. 329. 635-638 (1987)

[Publications] K.Sutoh: "Spatial proximity of the glycine-rich loop and the SH2 thiol in myosin subfragment 1." Biochemistry. 27. 2964-2969 (1988)

[Publications] M.Suzuki: "Packing of DNA in Cricket Sperm.A Compact Mode of DNA Packaging." J.Mol.Biol.204. 653-661 (1988)

[Publications] K.Hirose: "Thin filaments of Rabbit Skeletal Muscle are in Helical Register." J.Mol.Biol.204. 797-801 (1988)

[Publications] K.Sutoh: "End-label fingerprintings show that an N-terminal segment of depactin participates in interaction with actin." Biochemistry. 28. 102-106 (1989)

[Publications] K.Sutoh: "End-labeling fingerprinting shows that the N- and C-termini of actin are in the contact site with gelsolin." Biochemistry. 28. 5269-5275 (1989)

[Publications] K.Sutoh: "Electron Microscopic Mapping of Myosin Head with Site-directed Antibodies." J.Mol.Biol.206. 357-363 (1989)

[Publications] K.Saeki: "New Method to Prepare single-Headed Heavy Meromyosin with High Purity and a High Yield." J.Biochem.106. 606-611 (1989)

[Publications] T.Wakabayashi: "Three-Dimensional Image Reconstruction from Electron Micrographs of Biological Macromolocules." J.Electron Microsc.38. S102-S104 (1989)

若林健之東京大学, 理学部, 教授 (90011717)