Sinard J H, Pollard T D
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1990 Mar 5;265(7):3654-60.
We have shown previously that Acanthamoeba myosin-II minifilaments assemble by three successive dimerization steps, forming, progressively, monomers, antiparallel dimers, antiparallel tetramers, and finally the full size octameric minifilament (Sinard, J. H., Stafford, W. F., and Pollard, T. D. (1989) J. Cell Biol. 109, 1537-1548). In the current study, we investigate the kinetics of the assembly of these minifilaments, initiating assembly by the rapid dilution of salt in a stopped-flow light scattering apparatus. The majority of the reaction is completed within 50 ms and is greater than 90% completed within 1 s. Assembly data over a greater than 6-fold myosin concentration range can be fit using the successive dimerization mechanism with a single set of rate constants. Second order rate constants for the initial steps in the assembly reaction exceed 10(8) M-1 s-1, and equilibrium dissociation constants predict a very low critical concentration, consistent with previous data. Other possible assembly mechanisms do not adequately fit all of the available data. Filament disassembly at 300 mM KCl is even more rapid, and there is both an increase in the dissociation rate constants and a decrease in the association rate constants with increasing KCl. Aggregation of minifilaments induced by Mg2+ is much slower and takes many minutes to reach equilibrium.
我们之前已经表明，棘阿米巴肌球蛋白-II微丝通过三个连续的二聚化步骤组装而成，逐步形成单体、反平行二聚体、反平行四聚体，最终形成完整大小的八聚体微丝（西纳德，J. H.，斯塔福德，W. F.，和波拉德，T. D.（1989年）《细胞生物学杂志》109卷，1537 - 1548页）。在当前的研究中，我们通过在停流光散射装置中快速稀释盐来引发组装，研究这些微丝组装的动力学。大部分反应在50毫秒内完成，在1秒内完成超过90%。在大于6倍肌球蛋白浓度范围内的组装数据可以用具有一组单一速率常数的连续二聚化机制来拟合。组装反应初始步骤的二级速率常数超过10⁸ M⁻¹ s⁻¹，平衡解离常数预测临界浓度非常低，这与先前的数据一致。其他可能的组装机制不能充分拟合所有可用数据。在300 mM KCl下的丝解聚甚至更快，并且随着KCl浓度增加，解离速率常数增加，缔合速率常数降低。Mg²⁺诱导的微丝聚集要慢得多，需要许多分钟才能达到平衡。
