Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase.
Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase.
复制标题
原肌球蛋白在平滑肌收缩中的作用：原肌球蛋白与肌动蛋白结合对肌球蛋白 ATP 酶的肌动蛋白激活的影响。
DOI：
10.1021/bi00357a067
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Chacko,S
中科院分区：
文献类型：
--
作者：
Miyata,H;Chacko,S
材料和方法如报道的（Chacko，1981; Chacko & Rosenfeld，1982），通过在Sepharose 4B-CL琼脂糖柱上的凝胶过滤，制备和纯化来自鸡砂囊的完全磷酸化的肌球蛋白，并进行微小的修改。将蛋白酶抑制剂加入到以下缓冲液中：洗涤和提取缓冲液、1 µ抗疼痛剂、1 mM EGTA、50 µ l反式-环氧-琥珀酰亮氨酰氨基（4-胍基）丁烷（E-64）（Y. Nonomura，University of Tokyo）、1 µ胃酶抑素A和0.1 mM苯甲磺酰氟（PMSF）;磷酸化缓冲液、2.0 µ抗疼痛剂、2.0 µ胃酶抑素A和0.2 mM PMSF;镁沉淀缓冲液、0.5 µ抗疼痛剂和0.5 µ胃酶抑素A。按照Ebashi（1985）的方法制备平滑肌肌动蛋白。根据Spudich和Watt（1971）的方法，去除了用该方法制备的肌动蛋白中存在的痕量原肌球蛋白。通过这种方法获得的F-肌动蛋白是高度粘稠的，不含原肌球蛋白。
Materials and MethodsFully phosphorylated myosin from chicken gizzard was prepared and purified by gel filtration on Sepharose 4B-CL agarose column as reported (Chacko, 1981; Chacko & Rosenfeld, 1982) with minor modifications. Protease inhibitors were added to the following buffers: wash and extraction buffer, 1 µ antipain, 1 mM EGTA, 50 µ l-íra «í-epoxy-succinylleucylamido (4-guanidino) butane (E-64)(a gift from Dr. Y. Nonomura, University of Tokyo), 1 µ pepstatin A, and 0.1 mM phenylmethanesulfonyl fluoride (PMSF); phos-phorylation buffer, 2.0 µ antipain, 2.0 µ pepstatin A, and 0.2 mM PMSF; magnesium precipitation buffer, 0.5 µ an-tipain and 0.5 µ pepstatin A.Smooth muscle actin was prepared following the method of Ebashi (1985). Trace amounts of tropomyosin present in the actin prepared by this method was removed according to Spudich and Watt (1971). The F-actin obtained by this method was highly viscous and free of tropomyosin.