Okuno Takashi, Yamada-Inagawa Tomoko, Karata Kiyonobu, Yamanaka Kunitoshi, Ogura Teru
Division of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 862-0976, Japan.
J Struct Biol. 2004 Apr-May;146(1-2):148-54. doi: 10.1016/j.jsb.2003.10.019.
We have established a fluorescence polarization assay system by which degradation of sigma32, a physiological substrate, by FtsH can be monitored spectrometrically. Using the system, it was found that an FtsH hexamer degrades approximately 0.5 molecules of Cy3-sigma32 per min at 42 degrees C and hydrolyzes approximately 140 ATP molecules during the degradation of a single molecule of Cy3-sigma32. Evidence also suggests that degradation of sigma32 proceeds from the N-terminus to the C-terminus. Although FtsH does not have a robust enough unfoldase activity to unfold a tightly folded proteins such as green fluorescent protein, it can unfold proteins with lower T(m)s such as glutathione S-transferase (T(m) = 52 degrees C).
我们建立了一种荧光偏振分析系统，通过该系统可以用光谱法监测FtsH对生理底物sigma32的降解。使用该系统发现，FtsH六聚体在42℃下每分钟降解约0.5个Cy3-sigma32分子，并且在单个Cy3-sigma32分子的降解过程中水解约140个ATP分子。有证据还表明，sigma32的降解是从N端向C端进行的。尽管FtsH没有足够强大的解折叠酶活性来展开诸如绿色荧光蛋白之类紧密折叠的蛋白质，但它可以展开具有较低解链温度（Tm）的蛋白质，如谷胱甘肽S-转移酶（Tm = 52℃）。
