PROTEOLYSIS OF SMOOTH-MUSCLE MYOSIN BY STAPHYLOCOCCUS-AUREUS PROTEASE - PREPARATION OF HEAVY-MEROMYOSIN AND SUBFRAGMENT-1 WITH INTACT 20 000-DALTON LIGHT-CHAINS
PROTEOLYSIS OF SMOOTH-MUSCLE MYOSIN BY STAPHYLOCOCCUS-AUREUS PROTEASE - PREPARATION OF HEAVY-MEROMYOSIN AND SUBFRAGMENT-1 WITH INTACT 20 000-DALTON LIGHT-CHAINS
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DOI：
10.1021/bi00330a038
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
HARTSHORNE, DJ
中科院分区：
文献类型：
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作者：
IKEBE, M;HARTSHORNE, DJ
金黄色葡萄球菌蛋白酶对[火鸡]胗肌球蛋白的蛋白水解产生重肌球蛋白和2万道尔顿L链完整的亚片段1，并提出了制备每种蛋白的条件。肌凝蛋白H链裂解产生亚片段1依赖于肌凝蛋白的构象。10S构象的肌凝蛋白蛋白水解主要产生H型肌凝蛋白，而6S构象的肌凝蛋白主要产生亚片段1和一些重型肌凝蛋白。有两个位点受肌凝蛋白构象的影响，它们位于。apprx。68,000和94,000道尔顿来自肌凝蛋白H链的n端。后一个位点被认为位于亚片段1-亚片段2连接处，该位点的裂解导致亚片段1的产生。重肌球蛋白和亚片段1磷酸化的时间过程可以用一个指数来拟合。肌动蛋白激活的Mg2+- atp酶活性被20,000道尔顿L链的磷酸化显著激活。从肌动蛋白对Mg2+- atp酶活性的依赖性可以得到以下值：对于磷酸化的重肌球蛋白，Vmax .apprx。5.6 s-1和Ka（肌动蛋白的表观解离常数）。2毫克/毫升;对于去磷酸化的重肌球蛋白，Vmax .apprx。0.2 s-1和Ka .apprx。7毫克/毫升。肌动蛋白激活的亚片段1的atp酶活性不受磷酸化影响，磷酸化和去磷酸化形式的Vmax和Ka分别为0.4 s-1和5 mg/ml。亚片段1的Mg2+-和Ca2+- atp酶活性与重肌球蛋白不同，并且亚片段1的Mg2+- atp酶活性不受磷酸化的影响。亚片段1的20,000道尔顿L链的磷酸化显然不是肌动蛋白激活的atp酶活性所必需的。磷酸化调控涉及肌凝蛋白头部与分子其他部分的相互作用，可能在亚片段2区域。
The proteolysis of [turkey] gizzard myosin by S. aureus protease produces both heavy meromyosin and subfragment 1 in which the 20,000-dalton L chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin H chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly H meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at .apprx. 68,000 and 94,000 daltons from the N-terminus of the myosin H chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20,000-dalton L chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax .apprx. 5.6 s-1 and Ka (the apparent dissociation constant for actin) .apprx. 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax .apprx. 0.2 s-1 and Ka .apprx. 7 mg/ml. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/ml, respectively. The Mg2+- and Ca2+-ATPase activities of subfragment 1 are distinct from those of heavy meromyosin, and the Mg2+-ATPase activity of subfragment 1 is not affected by phosphorylation. The phosphorylation of the 20,000-dalton L chain of subfragment 1 apparently is not required for actin-activated ATPase activity. Regulation via phosphorylation involves the interaction of the myosin heads with other parts of the molecule, possibly in the subfragment 2 region.