Kalabokis V N, Vibert P, York M L, Szent-Györgyi A G
Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
J Biol Chem. 1996 Oct 25;271(43):26779-82. doi: 10.1074/jbc.271.43.26779.
Single-headed scallop myosin (shM) was prepared by papain digestion of filamentous scallop myosin and purified by hydrophobic interaction chromatography. The shM preparation consisted of equimolar amounts of polypeptides corresponding to an intact heavy chain, rod chain, essential light chain, and regulatory light chain. In electron micrographs the shape of shM showed the presence of a single head domain to which a normal looking rod was attached. Myosin and shM bound Ca2+ with association constants of 5 x 10(6) and 11 x 10(6) M-1, respectively. The ATPase activity of shM was activated about 3-fold by Ca2+. Both heads of myosin and shM had comparable ATPase activities in the presence of Ca2+. The activation of the ATPase activity of single-headed scallop myosin by Ca2+ paralleled closely the Ca2+ binding, in sharp contrast to the activation of intact myosin by Ca2+, which is highly cooperative. Single turnover experiments of myosin with radioactive ATP gave a half-life for the ATPase cycle of approximately 3 min in the presence of EGTA, whereas that of single-headed myosin was shorter than approximately 30 s, which was the resolution time of these measurements. The results suggest that the presence of two heads, as well as the attachment of the head to the coiled coil rod, contribute to the regulation of scallop myosin by Ca2+.
单头扇贝肌球蛋白（shM）通过用木瓜蛋白酶消化丝状扇贝肌球蛋白制备，并通过疏水相互作用色谱法纯化。shM制剂由等摩尔量的对应于完整重链、杆链、必需轻链和调节轻链的多肽组成。在电子显微镜照片中，shM的形状显示存在一个附着有正常外观杆的单头结构域。肌球蛋白和shM结合Ca2+的缔合常数分别为5×10(6)和11×10(6) M-1。shM的ATP酶活性被Ca2+激活约3倍。在存在Ca2+的情况下，肌球蛋白和shM的两个头部具有相当的ATP酶活性。单头扇贝肌球蛋白的ATP酶活性被Ca2+激活与Ca2+结合密切平行，这与完整肌球蛋白被Ca2+激活形成鲜明对比，完整肌球蛋白的激活是高度协同的。用放射性ATP对肌球蛋白进行单周转实验，在存在EGTA的情况下，ATP酶循环的半衰期约为3分钟，而单头肌球蛋白的半衰期短于约30秒，这是这些测量的分辨率时间。结果表明，两个头部的存在以及头部与卷曲螺旋杆的附着有助于Ca2+对扇贝肌球蛋白的调节。
