An enzyme-probe method to detect structural changes in the myosin rod.
An enzyme-probe method to detect structural changes in the myosin rod.
复制标题
检测肌球蛋白杆结构变化的酶探针方法。
DOI：
10.1016/0022-2836(84)90402-9
复制
发表时间：
1984
影响因子：
5.6
通讯作者：
Harrington,WF
中科院分区：
文献类型：
--
作者：
Ueno,H;Harrington,WF
通过跟踪蛋白水解消化速率常数的变化，研究了兔肌球蛋白杆α-螺旋、卷曲螺旋结构内局部熔化的温度依赖性。通过十二烷基硫酸钠/聚丙烯酰胺凝胶上消化产物的电泳，监测了在5至40 °C（pH 7，I= 0.5）的温度范围内，三种不同酶（α-糜蛋白酶、胰蛋白酶和木瓜蛋白酶）对棒的断裂动力学。所有的速率常数进行了校正的内在的温度依赖性的酶与模型基板比较。从三种酶探针的结果是相似的，显示在两个不同的阶段发生在杆内的局部熔化。在5 ° C至25 °C的温度下，熔化被限制在靠近轻解肌球蛋白/重解肌球蛋白连接处的杆状结构的受限区段。在25 ° C和40 °C之间的温度下，从在该区域观察到的广谱裂解位点判断，在连接和短亚片段-2片段（铰链结构域）之间裂解的较宽的棒状体片段似乎正在熔化。结果进行了比较，从其他物理化学方法，测量铰链或开放的螺旋线圈结构的杆。
The temperature-dependence of local melting within the α-helical, coiled-coil structure of rabbit myosin rod has been investigated by following changes in the rate constants of proteolytic digestion. The kinetics of fragmentation of the rod by three different enzymes (α-chymotrypsin, trypsin and papain) over the temperature range 5 to 40 °C (pH 7,I= 0.5) has been monitored by electrophoresis of the digestion products on sodium dodecyl sulfate/ polyacrylamide gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzyme by comparison with model substrates. Results from the three enzyme-probes are similar in showing that local melting within the rod occurs in two distinct stages. At temperatures between 5 and 25 °C, melting is confined to a restricted segment of the rod structure near the light meromyosin/heavy meromyosin junction. At temperatures between 25 and 40 °C, a wider segment of the rod lysing between the junction and the short subfragment-2 segment (the hinge domain) appears to be melting, judging from the broad spectrum of cleavage sites observed in this region. Results are compared with those from other physicochemical methods that measure the hinging or opening of the coiled-coil structure of the rod.