Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin.
Exchange of the fluorescence-labeled 20,000-dalton light chain of smooth muscle myosin.
复制标题
平滑肌肌球蛋白荧光标记的 20,000 道尔顿轻链的交换。
DOI：
10.1021/bi00103a022
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Ikebe,M
中科院分区：
文献类型：
--
作者：
Morita,J;Takashi,R;Ikebe,M
生理学和生物物理学系，凯斯西储大学，克利夫兰，俄亥俄州44106，贝勒研究所，贝勒大学医学中心，达拉斯，得克萨斯州75226接收日期：1990年11月7日;修订后的手册，接收日期：1991年4月16日摘要：在40 ℃下，在含有0.5M NaCl和10 mM EDTA的溶液中，将平滑肌肌球蛋白的20000-道尔顿轻链与外源性轻链交换。在上述条件下，轻链在30 min内几乎完全交换。在低于37 ℃或Mg ~（2+）浓度高于10 μ M时，交换被明显抑制。20000-道尔顿轻链用5-[[2-[（碘乙酰基）氨基]乙基]氨基-萘-1-磺酸（1，5-IAEDANS）选择性标记单个巯基（Cys-108）。将标记的轻链化学计量地交换到肌球蛋白中，并用作探针来研究平滑肌肌球蛋白的构象。所得的肌球蛋白杂交体显示出与对照组（未处理的肌球蛋白）几乎相同的酶性质;即肌动蛋白激活的ATP酶活性依赖于肌球蛋白轻链激酶催化的20000-道尔顿轻链磷酸化，与ATP酶变化相关的肌球蛋白10 S-6S构象转变也受光-链磷酸化或通过离子强度的变化。通过改变温度进行稳态荧光各向异性测量。构建Perrin-Weber图是为了获得关于探针的平均旋转移动性的信息，并估计AEDANS-肌球蛋白头的旋转相关时间。发现20 000-道尔顿轻链上的荧光探针相当不动，如其极限各向异性（A0= 0.33）所示。在20 ℃下，附着的AEDANS的旋转相关时间从AEDANS-20 000-道尔顿轻链的8.7 ns增加到肌球蛋白亚片段1（S1）时的16.8 ns，但该值明显小于S-1大小的无水刚性球的计算值，这表明S1的轻链结合位点是部分柔性的和/或轻链本身相对于S1结构的其余部分具有相当大的运动自由度。AE-DANS标记的轻链与6S肌球蛋白的结合仅导致旋转相关时间比S1的旋转相关时间略微增加，而AE-DANS标记的轻链与10 S肌球蛋白结合的旋转相关时间显著大于用6S肌球蛋白获得的旋转相关时间。类似地，去磷酸化的H-裂肌球蛋白在低离子强度（0.03 Mkc ~ 1）下的旋转相关时间显著大于在高离子强度（0.4 Mkc ~ 1）下的旋转相关时间。这些结果表明，平滑肌肌球蛋白从6S到10 S的构象转变引起了位于肌球蛋白头-杆连接处的20000道尔顿轻链结合位点的构象变化。
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, and Baylor Research Institute, Baylor University Medical Center, Dallas, Texas 75226 Received November 7, 1990; Revised Manuscript Received April 16, 1991 abstract: The 20 000-dalton light chain of smooth muscle myosin was exchanged with exogenous light chain in a solution containing 0.5 M NaCl and 10 mM EDTA at 40 C. The light chain was almost completely exchanged within 30 min under theabove conditions. The exchange was markedly inhibited either below 37 C or in the presence of Mg2+ concentrations higher than 10/tM. The 20000-dalton light chain was selectively labeled of a single thiol (Cys-108) with 5-[[2-[(iodoacetyl) amino] ethyl] amino-naphthalene-1-sulfonic acid (1, 5-IAEDANS). The labeled light chain was exchanged stoichiometrically into myosinand was used as a probe to investigate the conformation of smooth muscle myosin. The resulting myosin hybrids showed enzymatic properties virtually identical with those of the control, untreated myosin; ie, actin-activated ATPase activity was dependent on the 20000-dalton light-chain phosphorylation catalyzed by myosin light chain kinase, and the 10S-6S conformational transition of myosin correlating with the changes in ATPase was also affectedeither by the light-chain phosphorylation or by the change in the ionic strength. Steady-state fluorescence antisotropy measurements were performed by varying the temperature. The Perrin-Weber plots were constructed in order to obtain information about the average rotational mobility of the probe and to estimate the rotational correlation time for the AEDANS-myosin head. The fluorescence probe on the 20 000-dalton light chain was found to be quite immobile as indicated by its limiting anisotropy (A0= 0.33). The rotational correlation time of attached AEDANS at 20 C increased from 8.7 ns in AEDANS-20 000-dalton light chain to 16.8 ns when incorporatedinto myosin subfragment 1 (Sl), but this value was significantly smaller than that calculated for an anhydrous, rigid sphere of the size of S-1, suggesting that the light-chain binding site of Sl is partially flexible and/or the light chain itself has considerable motional freedom with respective to the rest of the Sl structure. The binding of AE-DANS-labeled light chainto 6S myosin resulted in only a slight increase in the rotational correlation time over that of Sl while the rotational correlation time of AEDANS-labeled light chain bound to 10S myosin was significantly larger than those obtained with 6S myosin. Analogously, the rotational correlation time of dephosphorylated H-meromyosin was significantly larger at low ionic strength (0.03 Mkc1) thanthat at high ionic strength (0.4 Mkc1). These results suggest that the conformational transition of smooth muscle myosin from 6 S to 10 S brings about a change in conformation of the 20 000-daltonlight-chain binding site of myosin which is located in the head-rodjunction of myosin.
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DOI：
10.1073/pnas.74.1.129
发表时间：
1977-01-01
影响因子：
11.1
作者：
CHACKO, S;CONTI, MA;ADELSTEIN, RS
通讯作者：
ADELSTEIN, RS
影响因子：
3.9
作者：
S. Driska;D. Hartshorne
通讯作者：
D. Hartshorne
与平滑肌和骨骼肌肌球蛋白相比，血小板肌球蛋白活性位点的表征。
影响因子：
3.9
作者：
I. Cohen;E. Kaminski;R. Lamed;A. Oplatka;A. Mühlrád
通讯作者：
A. Mühlrád
[30] 平滑肌肌球蛋白轻链激酶
DOI：
--
发表时间：
1983
期刊：
影响因子：
--
作者：
M. Walsh;S. Hinkins;R. Da̧browska;D. Hartshorne
通讯作者：
D. Hartshorne
平滑肌肌球蛋白的构象依赖性蛋白水解。
DOI：
--
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Ikebe,M;Hartshorne,DJ
通讯作者：
Hartshorne,DJ