Okamoto Y, Okamoto M, Sekine T
J Biochem. 1980 Aug;88(2):361-71. doi: 10.1093/oxfordjournals.jbchem.a132981.
The locations of ATP-induced structural change in gizzard myosin were examined by analyzing the changes in the chymotryptic fragmentation pattern. From the time course of fragmentation, and by fractionation of the produced fragments and detection of the masked amino terminal fragment, the original positions of the six different fragments in the parent myosin molecule were assigned. A reconstituted model of myosin based on the above assignment showed the presence of three cleavable sites in the heavy chain of myosin. ATP accelerated the cleavage at site 1, 5 K daltons from the masked amino terminus, while it inhibited cleavage at site 2, 71 K daltons from the N terminus. These opposite effects of ATP on sites 1 and 2 were remarkable, but ATP had no significant effect on cleavage at site 3, 100 K daltons from the carboxyl terminus. These results indicate that two distant regions in the myosin head, 66 K daltons apart in the primary sequence, were exposed or buried with the progress of the ATPase reaction. In addition, prolonged chymotryptic digestion failed to produce any subfragment-1, irrespective of the presence or absence of divalent cations in the digestion medium, but produced a protease-resistant and relatively long (100 K daltons) light meromyosin. This suggests a distinctive feature of the neck and hinge regions in gizzard myosin.
通过分析胰凝乳蛋白酶裂解模式的变化，研究了ATP诱导的肌胃肌球蛋白结构变化的位置。根据裂解的时间进程，对产生的片段进行分级分离，并检测被掩盖的氨基末端片段，确定了亲本肌球蛋白分子中六个不同片段的原始位置。基于上述定位构建的肌球蛋白重组模型显示，肌球蛋白重链中存在三个可裂解位点。ATP加速了距被掩盖氨基末端5千道尔顿处位点1的裂解，同时抑制了距N末端71千道尔顿处位点2的裂解。ATP对位点1和位点2的这些相反作用很显著，但ATP对距羧基末端100千道尔顿处位点3的裂解没有显著影响。这些结果表明，在肌球蛋白头部，一级序列中相距66千道尔顿的两个遥远区域随着ATP酶反应的进行而暴露或掩埋。此外，无论消化介质中是否存在二价阳离子，长时间的胰凝乳蛋白酶消化都未能产生任何亚片段-1，但产生了一种抗蛋白酶且相对较长（100千道尔顿）的轻酶解肌球蛋白。这表明了肌胃肌球蛋白颈部和铰链区的一个独特特征。
