A Rapid-freeze Electron Microscopy in Biological Materials

生物材料中的快速冷冻电子显微镜

• 批准号: 01480541
• 负责人: TSUKITA Shoichiro
• 金额: $ 3.84万
• 依托单位: Okazaki National Research Institutes (1990)Tokyo Metropolitan Organization for Medical Research (1989)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480541/
• 关键词: Caged compounds; Electron Microscope; Myosin; Rapid freezing; Liquid helium; ATP; 急速凍結法; 骨格筋; カルシウム; 蛋白質

The interaction between myosin subfragment 1(Sl)and actin filaments after the photolysis of P^3-1-(2-nitrophenyl)ethyl ester of ATP(caged ATP)was analyzed using a newly-developed freezing system with liquid helium. Actin and Sl(100g mu each)formed a rope-like double helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. A UV flash with a half duration time of 0.3 ms photoreleased -0.8 mM of ATP. At 15 ms after photolysis, the rope-like double helix was partially disintegrated. S1 on actin filaments gradually decreased in number up to 35 ms after photolysis and their association was in a somewhat cooperative manner. No more changes of Sl attached on actin filaments were detected from 35 to 200 ms. At 1 min after photolysis, ATP was consumed and the rope-like double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most Sl observed on actinfilaments at 15-200 ms are so called "weakly-bound Sl"(SL. ATP or Sl. ADP. Pi)and that the gradual decrease of the weakly-bound Sl during 15-35 ms is due to the transition from the rapid equilibrium of actin. Sl. ATP/SI. ATP to that of actin. Sl. ADP. Pi/Sl. ADP. Pi.

用一种新研制的液氦冷冻系统研究了P^3-1-（2-硝基苯基）乙基ATP（笼状ATP）光解后肌球蛋白亚片段1（S1）与肌动蛋白丝的相互作用。肌动蛋白和S1（各100 μ g）在室温下，在5 mM笼状ATP存在下形成具有僵硬特征的绳状双螺旋。半持续时间为0.3 ms的UV闪光光释放约0.8 mM ATP。在光解后15 ms，绳状双螺旋部分解体。S1的肌动蛋白丝的数量逐渐减少到35毫秒后，光解和他们的协会是在某种程度上合作的方式。在35 ~ 200 ms内，没有发现更多的S1蛋白附着在肌动蛋白丝上的变化。在光解后1 min，ATP被消耗，绳状双螺旋被重新形成。考虑到最近对肌动球蛋白动力学的分析，我们得出结论，在15-200 ms在肌动丝上观察到的大多数Sl是所谓的“弱结合Sl”（SL。ATP或SI。ADP Pi），弱结合的Sl在15-35 ms内逐渐减少是由于肌动蛋白从快速平衡状态的转变。SL. ATP/SI。ATP与肌动蛋白之比。SL. ADPπ/Sl. ADP Pi.

项目成果

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Nishikawa,S.：“粘附连接蛋白沿着大鼠门牙分泌性成釉细胞之间可能的滑动界面定位。”

Sato,N.: "Radixin,a barbedーendーcapping actinーmodulating protein,is concentrated at the cleavage furrow during cytokinesis." J.Cell Biol.

Sato, N.：“Radixin 是一种带倒刺末端的肌动蛋白调节蛋白，在胞质分裂过程中集中在裂解沟中。”

月田早智子,檜枝洋記,月田承一郎: "A new 82-kD barbed end-capping protein(redixin)localized in the cell-to-cell adherens junction:purification and characterization" J.Cell Biol.108. 2369-2382 (1989)

Sachiko Tsukita、Hiroki Hinoeda、Shoichiro Tsukita：“一种位于细胞与细胞粘附连接处的新型 82-kD 带刺封端蛋白（redixin）：纯化和表征”J.Cell Biol.108（1989）。 ）

Tsukita,Sh.,Itoh,M.,Tsukita,Sa.: "A new 400 kD-protein from isolated adherens junctions:Its localization at the undercoat of adherens junctions and at microfilament bundles such as stress fibers and circumferential bundles." J.Cell Biol.109. 2905-2915 (19

Tsukita,Sh.、Itoh,M.、Tsukita,Sa.：“一种来自分离的粘附连接的新 400 kD 蛋白质：其定位于粘附连接的底毛和微丝束，例如应力纤维和周向束。”

Tsukita, Sh., Tsukita, Sa. and Nagafuchi, A.: "The undercoat of adherens junctions : A key specialized structure in organogenesis and carcinogensis." Cell Struct. Funct.15. 7-12 (1990)

月田，Sh.，月田，Sa。