Carlier M F, Pantaloni D
Biochemistry. 1986 Dec 2;25(24):7789-92. doi: 10.1021/bi00372a001.
The sequence of reactions involved in the polymerization of ATP-actin and accompanying hydrolysis of ATP has been investigated by using a new glass-fiber filter assay. The assay allows the rapid separation of filaments from monomeric actin, and therefore the straightforward identification of the nucleotide bound to F-actin in the time course of polymerization, using double-labeled [gamma-32P,3H]ATP. The data bring a direct confirmation of the existence of the previously proposed ATP-F-actin intermediate in the time course of polymerization. Moreover, comparison of the hydrolyzed ATP (i.e., acid-labile [32P]Pi) and of 32P bound to F-actin provides direct evidence for the second intermediate ADP-Pi-F-actin in the polymerization process. This latter species is the major transient in the polymerization of ATP-actin, its lifetime being of the order of minutes.
利用一种新的玻璃纤维滤膜分析法，研究了ATP - 肌动蛋白聚合过程中涉及的反应序列以及伴随的ATP水解反应。该分析法能够快速从单体肌动蛋白中分离出细丝，因此，使用双标记的[γ-32P,3H]ATP，能够在聚合反应的时间进程中直接鉴定与F - 肌动蛋白结合的核苷酸。这些数据直接证实了在聚合反应时间进程中先前提出的ATP - F - 肌动蛋白中间体的存在。此外，对水解的ATP（即酸不稳定的[32P]Pi）和与F - 肌动蛋白结合的32P进行比较，为聚合过程中的第二种中间体ADP - Pi - F - 肌动蛋白提供了直接证据。后一种物质是ATP - 肌动蛋白聚合过程中的主要瞬态物质，其寿命约为几分钟。
