Johara M, Toyoshima Y Y, Ishijima A, Kojima H, Yanagida T, Sutoh K
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Japan.
Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2127-31. doi: 10.1073/pnas.90.6.2127.
Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin filament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/D363/E364 are not essential for ATP-driven sliding and force generation.
通过定点诱变将盘基网柄菌肌动蛋白亚结构域1中的氨基酸残基D24/D25、E99/E100、E360/E361和D363/E364替换为组氨酸残基。突变型肌动蛋白在盘基网柄菌细胞中表达并纯化至同质。测量突变型肌动蛋白丝在附着于玻璃表面的重酶解肌球蛋白上的滑动运动，以评估突变对肌动蛋白运动性的影响。对于两个C端突变体，还测量了单根肌动蛋白丝和肌球蛋白产生的力。这些测量结果表明，D24/D25和E99/E100都参与ATP驱动的滑动，而E360/E361/D363/E364对于ATP驱动的滑动和力的产生不是必需的。
