Reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with myosin subfragment one: evidence for formation of a single protein disulfide with trapping of metal nucleotide at the active site.
Reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with myosin subfragment one: evidence for formation of a single protein disulfide with trapping of metal nucleotide at the active site.
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5,5-二硫双（2-硝基苯甲酸）与肌球蛋白亚片段一的反应：形成单一蛋白质二硫化物并在活性位点捕获金属核苷酸的证据。
DOI：
10.1021/bi00549a030
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
R. Yount
中科院分区：
文献类型：
--
作者：
J. A. Wells;R. Yount
James A.Wells和Ralph G.Young摘要：在0℃、pH 8.0的条件下，用双摩尔过量的5，5‘-二硫双(2-硝基苯甲酸)(DTNB)在MgADP存在下处理兔骨骼肌球蛋白乳糜蛋白酶亚段1(SF_1)，可使ATPase完全激活。失活分两个阶段发生，最终导致三个SH基团的修饰。在第一阶段，DTNB与两个SH基团发生快速反应(f/2~10min)，导致Ca~(2+)-ATPase的激活和K~+-EDTA-ATPase的失活。这些快速反应的SH基团中只有一个被认为是活动所必需的。在第二阶段(/，/2~2 h)，硫代硝基苯甲酸(TNB)封闭了一个快速反应的SH基团，被另一个被认为是关键的硫醇SH-2取代，形成了半胱氨酸二硫键。后一种反应导致所有的Ca~(2+)-ATPase活性丧失，并伴随着在活性部位(II/2，0ff速率~6天)捕捉到MgADP。用二硫代赤藓糖醇处理完全灭活的SF，减少了二硫键，减少了剩余的TNB-SF，混合二硫键，并释放了镁ADP，所有ATPase活性完全恢复。在该反应中，剩余的TNB为
James A. Wells and Ralph G. Yount* abstract; Treatment of rabbit skeletal myosin chymotryptic subfragment one (SF,) in the presence of MgADP with a twofold molar excess of 5, 5'-dithiobis (2-nitrobenzoicacid)(DTNB) at 0 C, pH 8.0, results in complete ATPase inac-tivation. Inactivation occurs in two phases that ultimately result in the modification of three SH groups. In the first phase there isa rapid reaction(f,/2~ 10 min) of DTNB with two SH groups which leads to activation of the Ca2+-ATPase and inactivationof the K+-EDTA-ATPase. Only one of these fast-reacting SH groups is believed essential for activity. In the second phase (/,/2~ 2 h), a thionitrobenzoic acid (TNB) group blocking one of the fast-reactingSH groups is displaced by a neighboring thiol believed to be the critical thiol called SH-2 to form a cystine disulfide bond. This latter reaction resulted in the loss of all Ca2+-ATPase activity with concom-itant trapping of MgADP at the active site (ii/2, 0ff rate~ 6 days). Treatment of fully inactivated SF, with dithioerythritol reduced the disulfide, reduced the remaining TNB-SF, mixed disulfide, and released MgADP with the full recovery of all ATPase activity. In this reaction the remaining TNB was