Miller L, Phillips M, Reisler E
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Eur J Biochem. 1988 May 16;174(1):23-9. doi: 10.1111/j.1432-1033.1988.tb14057.x.
Solution properties of skeletal muscle actin, modified at lysine-61 with fluorescein isothiocyanate (FITC) [Burtnick, L.D. (1984) Biochim. Biophys. Acta 791, 57-62], were re-examined in this work by light scattering, analytical ultracentrifugation, fluorescence, electron microscopy and myosin ATPase activity measurements. Fluorescence measurements using trace amounts of actin labeled with N-(1-pyrenyl)iodoacetamide showed that the FITC modification inhibited but did not block completely the polymerization of actin by KCl and MgCl2. Sedimentation velocity runs of FITC-actin, incubated with 100 mM KCl and 2 mM MgCl2, revealed the presence in these solutions of polymeric, oligomeric and monomeric species. The critical concentration for FITC-actin polymerization under these conditions was 12 microM. As judged by electron microscopy, FITC-actin polymers were similar to but generally shorter than standard F-actin filaments. Light scattering measurements indicated that FITC modification inhibited also the polymerization of actin by myosin subfragment 1 (S1) but the resulting complexes were indistinguishable from standard, decorated actin filaments. MgATPase measurements showed that FITC-actin, polymerized by preincubation with S1, activated the MgATPase activity of S1 while the monomeric labeled protein did not. Thus, in analogy to native actin, the activating function of FITC-actin depended on the formation of actin filaments. Results presented in this study suggest that the region around lysine-61 of actin plays an important role in actin-actin contact and is less crucial to actomyosin interaction.
本研究通过光散射、分析超速离心、荧光、电子显微镜和肌球蛋白ATP酶活性测量等方法，重新检测了用异硫氰酸荧光素（FITC）修饰赖氨酸-61的骨骼肌肌动蛋白的溶液性质[Burtnick, L.D.（1984年），《生物化学与生物物理学报》791, 57 - 62]。使用微量N-(1-芘基)碘乙酰胺标记的肌动蛋白进行荧光测量表明，FITC修饰抑制但未完全阻断KCl和MgCl2诱导的肌动蛋白聚合。在含有100 mM KCl和2 mM MgCl2的条件下孵育FITC - 肌动蛋白并进行沉降速度实验，结果显示这些溶液中存在聚合物、寡聚体和单体形式。在这些条件下，FITC - 肌动蛋白聚合的临界浓度为12 μM。通过电子显微镜判断，FITC - 肌动蛋白聚合物与标准F - 肌动蛋白丝相似，但通常较短。光散射测量表明，FITC修饰也抑制了肌动蛋白与肌球蛋白亚片段1（S1）的聚合，但形成的复合物与标准的、被装饰的肌动蛋白丝无法区分。MgATP酶活性测量表明，预先与S1孵育聚合的FITC - 肌动蛋白可激活S1的MgATP酶活性，而单体标记蛋白则不能。因此，与天然肌动蛋白类似，FITC - 肌动蛋白的激活功能取决于肌动蛋白丝的形成。本研究结果表明，肌动蛋白赖氨酸-61周围区域在肌动蛋白 - 肌动蛋白接触中起重要作用，而对肌动球蛋白相互作用的重要性较低。
