Yamamoto K, Sekine T
J Biochem. 1986 Jan;99(1):199-206. doi: 10.1093/oxfordjournals.jbchem.a135460.
To elucidate the difference between subfragment-1 and heavy meromyosin in their interaction with F-actin, we used limited tryptic digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The binding of actin to subfragment-1 lowers the susceptibility of the 50K-20K junction of its heavy chain to tryptic digestion. At a molar ratio of one actin to one subfragment-1, all the sites were gradually cleaved by trypsin whereas the sites were completely protected in the presence of a 2-fold molar excess of actin over subfragment-1. In the case of heavy meromyosin, nearly half of the sites were protected completely by the presence of an equimolar amount of actin to its heads suggesting that the two heads of heavy meromyosin bound actin in a different manner. The rate of the cross-linking reaction between subfragment-1 heavy chain and actin with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide also depended on the molar ratio of actin to subfragment-1. The rate was maximum at a molar ratio of about 5 actin to 1 subfragment-1. When heavy meromyosin was cross-linked to actin, the maximum rate was observed at a molar ratio of about 3 actin to 1 heavy meromyosin head, the level being about 60% that for subfragment-1 and actin. It was suggested that the presence of the subfragment-2 portion of heavy meromyosin caused these differences by restricting the motion of the two heads.
为了阐明肌动蛋白亚片段1（subfragment-1）和重酶解肌球蛋白（heavy meromyosin）在与F-肌动蛋白相互作用方面的差异，我们采用了有限胰蛋白酶消化法以及与1-乙基-3-[3-(二甲氨基)丙基]碳二亚胺进行交联的方法。肌动蛋白与肌动蛋白亚片段1的结合降低了其重链50K-20K连接处对胰蛋白酶消化的敏感性。在肌动蛋白与肌动蛋白亚片段1的摩尔比为1:1时，所有位点会逐渐被胰蛋白酶切割，而当肌动蛋白的摩尔量超过肌动蛋白亚片段1两倍时，这些位点则受到完全保护。对于重酶解肌球蛋白而言，当肌动蛋白与其头部的摩尔量相等时，近一半的位点受到完全保护，这表明重酶解肌球蛋白的两个头部以不同方式结合肌动蛋白。肌动蛋白亚片段1重链与肌动蛋白通过1-乙基-3-[3-(二甲氨基)丙基]碳二亚胺进行交联反应的速率也取决于肌动蛋白与肌动蛋白亚片段1的摩尔比。在肌动蛋白与肌动蛋白亚片段1的摩尔比约为5:1时，反应速率达到最大值。当重酶解肌球蛋白与肌动蛋白交联时，在肌动蛋白与重酶解肌球蛋白头部的摩尔比约为3:1时观察到最大反应速率，该水平约为肌动蛋白亚片段1与肌动蛋白反应速率的60%。有人提出，重酶解肌球蛋白的亚片段2部分的存在通过限制两个头部的运动导致了这些差异。
