Characterization of calponin binding to actin.
Characterization of calponin binding to actin.
复制标题
钙调蛋白与肌动蛋白结合的表征。
DOI：
10.1021/bi00037a026
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Chalovich,JM
中科院分区：
文献类型：
--
作者：
Lu,FW;Freedman,MV;Chalovich,JM
材料和方法 骨骼肌肌球蛋白是从兔子的背部和腿部肌肉中制备的（Kielley & Harrington，1960）。 S-11通过用胰凝乳蛋白酶消化肌球蛋白来制备，并且通过DEAE-Sephacel色谱法分离A1和A2同工酶(Weeds & Taylor，1975)。采用 Spudich 和 Watt (1971) 的方法（经 Eisenberg 和 Kielley (1972) 修改）从兔背部和腿部肌肉中分离出骨骼肌动蛋白。按照 Strzelecka-Golaszewaka 等人 (1980) 的描述，从新鲜鸡肫中分离平滑肌肌动蛋白，并通过 AcA-54 凝胶色谱 (Spectrum) 进一步纯化。按照 Bretscher (1984) 的描述，原肌球蛋白是从火鸡砂囊中制备的。采用Yazawa等人(1980)的方法从猪脑中纯化钙调蛋白。如前所述（Velaz 等，1989），通过对 Bretscher（1984）的方法进行修改，从火鸡砂囊中纯化卡尔德斯蒙。平滑肌α-肌动蛋白是 Keith Burridge 博士（北卡罗来纳大学教堂山分校）的礼物。钙调蛋白是通过 Abe 等人 (1990) 描述的方法的改进来制备的。简而言之，将新鲜火鸡胗清洗、切碎，并在 Polytron 匀浆器的中间设置中均质化，加入 3 体积的缓冲液 A（50 mM 咪唑-HCl，pH 6.9，300 mM KCl，1 mM 二硫苏糖醇，10 pg/mL 亮肽素，0.5 mM 苯甲磺酰氟，50 pgj mL 大豆）胰蛋白酶抑制剂）。将匀浆在沸水浴中加热8-10分钟，并在冰上冷却
MATERIALS AND METHODSSkeletal muscle myosin was prepared from the back and leg muscle of rabbits (Kielley & Harrington, 1960). S-l1 was prepared from digestion of myosin with chymotrypsin, and the Al and A2 isozymes were separated by DEAE-Sephacel chromatography (Weeds & Taylor, 1975). Skeletal actin was isolated from rabbit back and leg muscles by the method of Spudich and Watt (1971) as modified by Eisenberg and Kielley (1972). Smooth muscle actin was isolated from fresh chicken gizzards as described by Strzelecka-Golaszewaka et al.(1980) and further purified by AcA-54 gel chromatography (Spectrum). Tropomyosin was prepared from turkey gizzards as described by Bretscher (1984). Calmodulin was purified from porcinebrains by the method of Yazawa et al.(1980). Caldesmon was purified from turkeygizzards by a modification of the method of Bretscher (1984) as described previously (Velaz et al., 1989). Smooth muscle a-actinin was a gift from Dr. Keith Burridge (University of North Carolina, Chapel Hill). Calponin was prepared by a modification of the method describedby Abe et al.(1990). Briefly, fresh turkey gizzards were cleaned, minced, and homogenized on the middle setting of a Polytron homogenizer in 3 volumes of buffer A (50 mM imidazole-HC1, pH 6.9, 300 mM KC1, 1 mM dithiothreitol, 10 pg/mL leupeptin, 0.5 mM phenylmethanesulfonyl fluoride, 50 pgj mL soybean trypsin inhibitor). The homogenate was heated in a boiling water bath for 8—10 min and cooled on ice for