Investigation, by cross-linking, of conformational changes in F-actin during its interactions with myosin.
Investigation, by cross-linking, of conformational changes in F-actin during its interactions with myosin.
复制标题
通过交联研究 F-肌动蛋白在与肌球蛋白相互作用过程中的构象变化。
DOI：
10.1021/bi00561a022
复制
发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Offer,G
中科院分区：
文献类型：
--
作者：
Knight,P;Offer,G
Materials and MethodsPreparation of Proteins. Actin was prepared by the methods of either Spudich & Watt (1971) or Hitchcock (1973) from an acetone powder of rabbit skeletal muscle (Straub, 1942; Katz & Hall, 1963). Twice-precipitated rabbit myosin was prepared by a method similar to that of Perry (1955). Sub-fragment-1 was prepared by papain digestion of myosin using either the method of Margossian & Lowey (1973a) or a modified version of the myofibril digestion method of Cooke (1972). For the latter, 300 mL of washed myofibrils in 0.1 Mkc1, 5 mM K2HP04, 5 mM KH2P04, and 1 mM MgCl2 at approximately 15 mg/mL total protein was digested at 25 C for 30 min by0. 011 mg/mL papain (Worthington; 19 units/mg dissolved in 10 mL of 10 mM cysteine and 10 mM EDTA, pH 6, prior to addition to fibrils). Digestion was terminated by 1 mM sodium iodoacetate. After three washes of the fibrils to remove peptide material and papain, the S-1 was released by theaddition of 1 mM magnesium pyro-phosphate with stirring for 20 min at 4 C. The fibrils were sedimented at 13000g, and the supernatant was recentrifuged at 78000g for 3 h to sediment the appreciablequantities of thin filaments that are released. The supernatant was dialyzed vs. 20 mM imidazole-HCl at pH 7.0 and chromatographed on a 2.5 X 25 cm column of DEAE-cellulose (Whatman DE-52) equilibrated with 20 mM imidazole-HCl, pH 7.0, and eluted with a linear gradient from 0 to 0.3 M NaCl in 1
