菠菜叶绿体CF_1-ATP合酶的分离纯化及其结构的原子力显微镜研究  

ISOLATION AND PURIFICATION OF SPINACH CHLOROPLAST CF_1-ATP AND ITS TOPOGRAPHY IMAGED BY ATOMIC FORCE MICROSCOPY

作　　者：朱华泽[1] 孙润广[1] 刘田[1] 徐平平[1] 

机构地区：[1]陕西师范大学物理学与信息技术学院生物物理与生物医学工程研究室,西安710062

出　　处：《生物物理学报》2008年第2期92-98,共7页Acta Biophysica Sinica

基　　金：国家自然科学基金项目(20572067);教育部科学技术研究重点项目(104167)~~

摘　　要：用纤维素DE-52柱层析和硫酸铵分级分离法快速分离、纯化获得CF1-ATP合酶,聚丙烯酰胺凝胶电泳技术对其纯度和凝胶内酶活性进行检测,研究了温度、pH值、盐浓度对CF1水解酶活性的影响,用原子力显微镜对CF1的形态结构进行了观察。结果发现,当pH值为8.0时,CF1可在0℃存放数天并保持其50%的活性,20%的甲醇溶液能够有效地保护酶分子的结构;实验还通过低温盐解作用获得了具有活性的亚基;用原子力显微镜观测显示CF1-ATP合酶分子平均大小约为18nm,按其直径大小可以分为三类,推测其分别对应着ATP合酶的三种状态,即空置态、疏松结合态和紧密结合态。CF1-ATPase was isolated and purified from spinach chloroplast by cellulose DE-52 chromatography and ammonium sulfate fractionation. Then its purity and in-gel activity were detected through PAGE. The effects of temperature, pH value and salt concentration on the activity of CF1-ATPase were also studied, and the topography of CF1-ATPase was observed by AFM. It was found that the activity kept 50% at 0℃ several days while pH value was 8.0, and that 20% methanol protected its molecule structure effectively. Besides, the authors obtained active subunits through incubating in 1 mol/L KC1 at 0℃. By AFM observation, the enzyme molecules were average 18 nm in diameter, and were sorted into 3 groups corresponding to three states （Omit state, Loose state and Tight state） of the enzyme respectively.

关 键 词：叶绿体 CF1-ATP合酶 AFM 水解活性 

分 类 号：Q615[生物学—生物物理学]