Barylko B, Tooth P, Kendrick-Jones J
Eur J Biochem. 1986 Jul 15;158(2):271-82. doi: 10.1111/j.1432-1033.1986.tb09747.x.
The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.
牛脑肌球蛋白的重链以及19 kDa和20 kDa的轻链能够被磷酸化。为了确定重链磷酸化的位点，首先在各种条件下用胰凝乳蛋白酶和木瓜蛋白酶对肌球蛋白进行消化，并对产生的片段进行鉴定。无论离子强度如何，即无论肌球蛋白是单体还是丝状，胰凝乳蛋白酶消化都会产生两个主要片段，分别为68 kDa和140 kDa；140 kDa的片段被木瓜蛋白酶进一步消化，产生一个120 kDa和一个23 kDa的片段。这些片段通过以下方法进行表征：(a) 使用125I标记的轻链的凝胶覆盖技术，该技术表明140 kDa和23 kDa的多肽含有轻链结合位点；(b) 使用在活性位点用[3H]UTP进行光亲和标记的肌球蛋白，该技术表明68 kDa的片段含有催化位点；(c) 使用旋转阴影和负染色技术的电子显微镜，该技术表明在胰凝乳蛋白酶消化后，肌球蛋白头部仍与尾部相连，而在木瓜蛋白酶消化后则观察到分离的头部和尾部。因此，源自140 kDa片段的120 kDa多肽是肌球蛋白的尾部，而含有催化位点的68 kDa片段和具有轻链结合位点的23 kDa片段形成肌球蛋白的头部(S1)部分。当用胰凝乳蛋白酶和木瓜蛋白酶消化[32P] - 磷酸化的脑肌球蛋白时，发现重链磷酸化位点位于重链C末端的一个5 kDa肽段中，即肌球蛋白尾部的末端。使用流体动力学和电子显微镜技术，即使在存在MgATP的情况下，也未观察到轻链或重链磷酸化对脑肌球蛋白丝稳定性有显著影响。脑肌球蛋白丝似乎比其他非肌肉肌球蛋白的丝更稳定。然而，轻链磷酸化确实对脑肌球蛋白的构象有影响，例如在存在MgATP的情况下，未磷酸化的肌球蛋白分子会被诱导折叠成非常紧密的折叠状态。
