Huang Y P, Kimura M, Tawada K
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
J Muscle Res Cell Motil. 1990 Aug;11(4):313-22. doi: 10.1007/BF01766669.
This paper describes a systematic study of crosslinking of skeletal muscle myosin subfragment-1 (S1) and heavy meromyosin (HMM) to F-actin in the rigor state with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). We followed the time courses of S1 or HMM head crosslinking at various actin:S1 or actin:HMM head molar ratios and the resulting superactivation of ATPase activity. The ATPase activity of the covalent complexes was measured at 0.5 M KCl, where the covalent complexes retain superactivated ATPase activity but the activity of uncrosslinked myosin heads is not activated by actin. S1 crosslinking was slowest at the actin:S1 molar ratio of 1:1, but faster at larger molar ratios, where more than 80% of added S1 could be crosslinked to actin. In spite of the dependence of crosslinking rate on actin:S1 ratio, there were two linear correlations between ATPase activity and the extent of S1 crosslinking to actin: one for S1 crosslinked to actin at actin:S1 molar ratios more than 2.7:1 and the other for S1 crosslinked at a molar ratio of 1:1. Extrapolation of the former correlation line to 100% crosslinked S1 gave an ATPase activity of 39 s-1 for actin-S1 covalent complex at 25 degrees C, whereas that of the other correlation line gave 21 s-1. The latter smaller activity suggests that the interface between actin and S1 in their rigor complexes at a molar ratio of 1:1 is different from that at molar ratios of more than 2.7:1. The acto-HMM crosslinking rate depended on the ratio of actin to HMM head, like that of S1 crosslinking to actin. The ATPase activity of crosslinked actin-HMM was, unlike that of actin-S1 covalent complexes, bell-shaped as a function of the crosslinked heads, but chymotryptic conversion of HMM to S1 in the covalent complexes made the bell-shaped characteristics disappear and increased the activity close to that of actin-S1 covalent complexes. These results indicate that some physical constraint imposed on myosin heads suppresses the actin-activated ATPase activity of HMM crosslinked to actin.
本文描述了一项关于在僵直状态下，用1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺（EDC）使骨骼肌肌球蛋白亚片段-1（S1）和重酶解肌球蛋白（HMM）与F-肌动蛋白交联的系统研究。我们追踪了在不同的肌动蛋白:S1或肌动蛋白:HMM头部摩尔比下S1或HMM头部交联的时间进程以及由此产生的ATP酶活性的超激活。共价复合物的ATP酶活性在0.5M KCl条件下测定，在此条件下共价复合物保留超激活的ATP酶活性，但未交联的肌球蛋白头部的活性不会被肌动蛋白激活。在肌动蛋白:S1摩尔比为1:1时，S1交联最慢，但在更大的摩尔比时更快，此时超过80%添加的S1可与肌动蛋白交联。尽管交联速率依赖于肌动蛋白:S1比例，但ATP酶活性与S1与肌动蛋白的交联程度之间存在两种线性关系：一种是在肌动蛋白:S1摩尔比大于2.7:1时与肌动蛋白交联的S1，另一种是在1:1摩尔比下交联的S1。将前一种相关线外推至100%交联的S1，得到25℃时肌动蛋白-S1共价复合物的ATP酶活性为39 s-1，而另一种相关线得到的活性为21 s-1。后一种较小的活性表明，在1:1摩尔比的僵直复合物中，肌动蛋白与S1之间的界面与大于2.7:1摩尔比时的界面不同。肌动蛋白-HMM交联速率依赖于肌动蛋白与HMM头部的比例，与S1与肌动蛋白的交联情况类似。与肌动蛋白-S1共价复合物不同，交联的肌动蛋白-HMM的ATP酶活性作为交联头部数量的函数呈钟形，但共价复合物中HMM经胰凝乳蛋白酶转化为S1后，钟形特征消失，活性增加至接近肌动蛋白-S1共价复合物的活性。这些结果表明，对肌球蛋白头部施加的一些物理限制抑制了与肌动蛋白交联的HMM的肌动蛋白激活的ATP酶活性。
