Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site.
Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site.
复制标题
脑肌球蛋白的蛋白水解片段和重链磷酸化位点的定位。
DOI：
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
J. Kendrick
中科院分区：
文献类型：
--
作者：
B. Baryłko;P. Tooth;J. Kendrick
牛脑肌球蛋白的重链以及19-kDa和20-kDa轻链可以被磷酸化。为了定位重链磷酸化位点，首先在多种条件下用胰凝乳蛋白酶和木瓜蛋白酶消化肌球蛋白，并鉴定由此产生的片段。无论离子强度如何，即肌球蛋白是单体还是丝状，胰凝乳蛋白酶消化都会产生 68 kDa 和 140 kDa 的两个主要片段； 140-kDa 片段进一步用木瓜蛋白酶消化，产生 120-kDa 和 23-kDa 片段。这些片段的特征在于：(a) 使用 125I 标记的轻链进行凝胶重叠技术，表明 140-kDa 和 23-kDa 多肽含有轻链结合位点； （b）使用在活性位点用[3H]UTP标记的肌球蛋白光亲和力，这表明68-kDa片段包含催化位点，以及（c）使用旋转阴影和负染色技术的电子显微镜，这表明在胰凝乳蛋白酶消化后，肌球蛋白头部仍然附着在尾部，而在木瓜蛋白酶消化时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，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5-kDa肽中，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，即使存在 MgATP，也没有观察到轻链或重链磷酸化对脑肌球蛋白丝稳定性的显着影响。脑肌球蛋白丝似乎比其他非肌肉肌球蛋白丝更稳定。然而，轻链磷酸化确实对脑肌球蛋白的构象有影响，例如在 MgATP 存在下，非磷酸化肌球蛋白分子被诱导折叠成非常紧凑的折叠状态。
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.
平滑肌肌球蛋白的构象状态。
DOI：
--
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Trybus,KM;Lowey,S
通讯作者：
Lowey,S
用 N-(4-叠氮基-2-硝基苯基)-2-氨乙基二磷酸进行光亲和标记后，鉴定骨骼肌球蛋白的活性位点肽。
DOI：
10.1073/pnas.82.6.1575
发表时间：
1985
影响因子：
11.1
作者：
Okamoto,Y;Yount,RG
通讯作者：
Yount,RG