Li W X, Schoenberg M
Laboratory of Physical Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Biochim Biophys Acta. 1998 Oct 5;1367(1-3):127-33. doi: 10.1016/s0005-2728(98)00138-8.
The finding of Barnett et al. (Biophys. J. 61 (1992) 358) that NPM-reacted crossbridge heads do not bind strongly to actin in rigor solution is not easily interpreted in terms of the solution studies of Xie and Schoenberg (Biochemistry 37 (1998) 8048) who found strong binding of NPM-reacted myosin subfragment-1 to actin in solutions devoid of MgATP. For this reason, the current work uses stiffness measurement to re-investigate the binding of rabbit skeletal muscle crossbridges to actin in rigor solution. It is found that NPM-reacted crossbridge heads bind strongly to actin in rigor solution providing one is extremely careful to reduce MgATP contamination to levels well below those that would have a detectable effect on unmodified fibers. The reason for this is that NPM-reacted crossbridge heads, which hydrolyze MgATP extremely slowly, are especially susceptible to contaminant MgATP. The new fiber results show a strong correlation with the solution results. A further manifestation of this correlation is that pPDM-reacted crossbridge heads are different from NPM-reacted ones in that, like in solution, they remain weakly binding to actin even at extremely low MgATP levels. The findings suggest that the covalent crosslinking of SH1 and SH2 by pPDM is likely playing a significant role in locking pPDM-reacted crossbridge heads in a weakly binding conformation.
巴尼特等人（《生物物理杂志》61卷（1992年）第358页）发现，在僵直溶液中，经NPM处理的横桥头部与肌动蛋白的结合并不紧密，这一发现很难根据谢和舍恩伯格的溶液研究结果（《生物化学》37卷（1998年）第8048页）来解释，后者发现经NPM处理的肌球蛋白亚片段-1在不含MgATP的溶液中与肌动蛋白有很强的结合。因此，当前的研究工作采用刚度测量方法，重新研究兔骨骼肌横桥在僵直溶液中与肌动蛋白的结合情况。研究发现，在僵直溶液中，经NPM处理的横桥头部与肌动蛋白有很强的结合，前提是要极其小心地将MgATP污染降低到远低于对未修饰纤维有可检测影响的水平。原因是经NPM处理的横桥头部水解MgATP的速度极慢，特别容易受到污染的MgATP的影响。新的纤维研究结果与溶液研究结果有很强的相关性。这种相关性的另一个表现是，经pPDM处理的横桥头部与经NPM处理的不同，就像在溶液中一样，即使在极低的MgATP水平下，它们与肌动蛋白的结合仍然很弱。这些发现表明，pPDM对SH1和SH2的共价交联可能在将经pPDM处理的横桥头部锁定在弱结合构象中发挥着重要作用。
