Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.
Both the 25-kDa and 50-kDa domains in myosin subfragment 1 are close to the reactive thiols.
复制标题
肌球蛋白亚片段 1 中的 25 kDa 和 50 kDa 结构域均接近反应性硫醇。
DOI：
10.1073/pnas.83.17.6392
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发表时间：
1986
影响因子：
11.1
通讯作者：
Wong,AG
中科院分区：
文献类型：
--
作者：
Lu,RC;Moo,L;Wong,AG
巯基特异性光活化试剂二苯甲酮-4-碘乙酰胺可掺入肌球蛋白亚片段1（S1）中，伴随着Ca ~（2+）-ATP酶活性的增加和K ~+-ATP酶活性的丧失，这是反应性巯基1（SH-1）被修饰时S1的特征性质。胰蛋白酶切割后，未光解样品的NaDodSO 4/聚丙烯酰胺凝胶电泳中发现了25-kDa、50-kDa和20-kDa片段，而光解样品中仅出现50-kDa片段和45-kDa片段，表明NH 2末端25-kDa片段通过SH-1与COOH末端20-kDa片段交联。当在Mg 2+和ATP或Mg 2+和腺苷5-[β，γ-亚氨基]三磷酸（ATP [NH]P）存在下进行光解时，还观察到70 kDa条带，可归因于交联（50 kDa + 20 kDa）物质。这表明核苷酸结合诱导的构象变化减少了50 kDa区域与SH-1上标记之间的距离。类似的结果时，标签和光解进行胰蛋白酶切口的S1，其中25-kDa，50-kDa，和20-kDa的片段举行在一起非共价。此外，当在Mg-ATP存在下进行二苯甲酮-4-碘乙酰胺标记时，这增加了另一种硫醇（推测为SH-2）的反应性，在不存在ATP的情况下光解时形成了45-kDa和70-kDa物质，表明SH-2接近50-kDa区域。更多的70-kDa的物种形成，在45-kDa的物种的费用，当在Mg-ATP的存在下进行光解。部分热变性优先减少反应性硫醇和50 kDa区域之间的交联。
The thiol-specific photoactivatable reagent benzophenone-4-iodoacetamide can be incorporated into myosin subfragment 1 (S1), accompanied by an increase of Ca2+-ATPase and the loss of K+-ATPase activities, a characteristic property of S1 when reactive sulfhydryl 1 (SH-1) is modified. After trypsin cleavage, 25-kDa, 50-kDa, and 20-kDa fragments were found upon NaDodSO4/polyacrylamide gel electrophoresis of the unphotolyzed sample, whereas only the 50-kDa fragment and a 45-kDa fragment appeared in the photolyzed sample, indicating that the NH2-terminal 25-kDa fragment was crosslinked to the COOH-terminal 20-kDa fragment via SH-1. When photolysis was carried out in the presence of Mg2+ and ATP or Mg2+ and adenosine 5-[beta, gamma-imido]triphosphate (AdoPP[NH]P), a 70-kDa band, attributable to a crosslinked (50 kDa + 20 kDa) species, was also observed. This suggests that the conformational change induced by nucleotide binding reduces the distance between the 50-kDa region and the label on SH-1. Similar results were obtained when labeling and photolysis were carried out on trypsin-nicked S1, in which the 25-kDa, 50-kDa, and 20-kDa fragments are held together noncovalently. Further, when labeling with benzophenone-4-iodoacetamide was carried out in the presence of Mg-ATP, which increases the reactivity of another thiol, presumably SH-2, both 45-kDa and 70-kDa species were formed upon photolysis in the absence of ATP, suggesting that SH-2 is close to the 50-kDa region. More of the 70-kDa species was formed, at the expense of the 45-kDa species, when photolysis was carried out in the presence of Mg-ATP. Partial heat denaturation preferentially reduced the crosslinking between the reactive thiols and the 50-kDa region.