Saeki K, Tokunaga M, Ting H A, Wakabayashi T
Department of Physics, Faculty of Science, University of Tokyo.
J Biochem. 1989 Oct;106(4):606-11. doi: 10.1093/oxfordjournals.jbchem.a122903.
We have developed a new method to prepare single-headed heavy meromyosin with high purity and a high yield. To examine whether the two heads on the same myosin molecule work cooperatively or not, it is important to prepare pure single-headed heavy meromyosin. Myosin was extracted from myofibrils treated with a solution containing CyDTA, a strong divalent cation chelator. CyDTA treatment was essential to the production of sHMM. Then such myosin was digested with chymotrypsin in the presence of divalent cations at high ionic strength. Crude sHMM was separated from double-headed HMM by affinity chromatography using an ADP-column. Contaminating S1 was removed by gel filtration. Heavy chain of sHMM obtained by the present method had no nick. Purified sHMM showed normal EDTA-ATPase and Ca-ATPase. It interacted with thin filament and its ATPase was activated by actin normally.
我们开发了一种新方法来制备高纯度、高产率的单头重酶解肌球蛋白。为了研究同一肌球蛋白分子上的两个头部是否协同工作，制备纯的单头重酶解肌球蛋白很重要。肌球蛋白从用含有强二价阳离子螯合剂CyDTA的溶液处理过的肌原纤维中提取。CyDTA处理对于sHMM的产生至关重要。然后在高离子强度的二价阳离子存在下，用胰凝乳蛋白酶消化这种肌球蛋白。通过使用ADP柱的亲和色谱法将粗制的sHMM与双头HMM分离。通过凝胶过滤去除污染的S1。通过本方法获得的sHMM重链没有切口。纯化的sHMM显示出正常的EDTA - ATP酶和Ca - ATP酶。它与细肌丝相互作用，并且其ATP酶被肌动蛋白正常激活。
