A Rapid-freeze Electron Microscopy in Biological Materials

点击翻译按钮获取中文摘要

The interaction between myosin subfragment 1(Sl)and actin filaments after the photolysis of P^3-1-(2-nitrophenyl)ethyl ester of ATP(caged ATP)was analyzed using a newly-developed freezing system with liquid helium. Actin and Sl(100g mu each)formed a rope-like double helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. A UV flash with a half duration time of 0.3 ms photoreleased -0.8 mM of ATP. At 15 ms after photolysis, the rope-like double helix was partially disintegrated. S1 on actin filaments gradually decreased in number up to 35 ms after photolysis and their association was in a somewhat cooperative manner. No more changes of Sl attached on actin filaments were detected from 35 to 200 ms. At 1 min after photolysis, ATP was consumed and the rope-like double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most Sl observed on actinfilaments at 15-200 ms are so called "weakly-bound Sl"(SL. ATP or Sl. ADP. Pi)and that the gradual decrease of the weakly-bound Sl during 15-35 ms is due to the transition from the rapid equilibrium of actin. Sl. ATP/SI. ATP to that of actin. Sl. ADP. Pi/Sl. ADP. Pi.