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Biochemistry

寄生性锥虫锥虫锥虫的 f1-atpase 的分离

Published: January 22, 2019
doi:
10.3791/58334
,
1Biology Centre,Czech Academy of Science, Institute of Parasitology, 2Faculty of Science,University of South Bohemia

Summary

该方案描述了从锥虫培养阶段的 f1-atpase 的纯化.该程序产生了高度纯、均匀和活性的复合物, 适用于结构和酶学研究。

Abstract

f1-atpase 是 f 型 atp 合成酶的膜外催化亚基, 是利用质子动力穿过生物膜产生三磷酸腺苷 (atp) 的酶.将完整的 f1-atpase 从其原生来源中分离出来是表征酶蛋白质组成、动力学参数和对抑制剂敏感性的必要前提.一种高度纯和均匀的f-1 atpase可用于结构研究, 从而深入了解 atp 合成和水解的分子机制。本文介绍了一种纯化非洲锥虫酶病原体—– ———————————————————————————————————f 1-atpase 从线粒体囊泡中分离出来, 线粒体囊泡是通过体外培养的锥体细胞的低渗裂解获得的.囊泡通过超声进行机械分裂, f1-atpase通过氯仿提取从线粒体内膜中释放。酶复合物通过连续阴离子交换和尺寸排除色谱进一步纯化。敏感质谱技术表明, 纯化后的复合物几乎没有任何蛋白质污染物, 因此是 x 射线晶体学或低温电子显微镜测定结构的合适材料。分离的f-1atpase 具有 atp 水解活性, 可通过 f 型 atp 合成酶的有效抑制剂–氮化钠完全抑制。在室温下, 纯化后的复合物保持稳定和活跃至少三天。硫酸铵沉淀用于长期储存。类似的程序已被用于纯化 f1-atpase 从哺乳动物和植物组织, 酵母, 或细菌.因此, 所提出的议定书可作为从其他生物体分离 f-1-atpase 的指南.

Introduction

f 型 atp 合成物是膜结合旋转多蛋白复合物, 它将质子在细菌、线粒体和叶绿体的能量传递膜上的移位与 atp 的形成结合在一起。atp 合成旋转机理的分子细节之所以知道, 主要是因为对纯化的细菌和线粒体 atp 合成酶及其亚配合物进行了结构研究1。f 型 atp 合酶被组织成膜固有的和膜外的摩尔。膜外源部分, 称为 f1-atpase,包含三个催化位点, 其中磷酸化的二磷酸腺苷 (adp) atp 或反向反应发生。f1-atpase 可以从膜固有的因子中实验释放, 同时保持其水解但不合成 atp 的能力.膜结合区, 称为 fo,介导蛋白质易位, 这驱动了酶的中心部分的旋转。f1和 f o扇区由中心和外围秸秆连接。

第一次尝试净化 f1-atpase从发芽的酵母和牛心脏线粒体可以追溯到20世纪60年代。这些协议使用提取的线粒体, 通过超声分解, 由铵或硫酸丙胺沉淀分解, 然后进行可选的色谱步骤和热处理2,3,4 ,5,6。氯仿的使用大大改善和简化了纯化, 很容易从线粒体膜碎片7中释放出 f1-atpase.然后用氯仿提取, 从各种动物、植物和细菌来源 (如大鼠肝脏8、玉米 9、金银花10和大肠杆菌)中提取 f1-atpase 11)。通过亲和或大小排除色谱法(sec) 进一步纯化氯仿释放的 f1-atpase, 产生了一种高度纯的蛋白质复合物, 适用于 x 射线晶体学的高分辨率结构测定。记录了牛心脏 12、13 和酿酒酵母14 的 f1-atpase 的结构.f-1-atpase 结构也是由难以培养的生物确定的, 因此, 最初的生物材料的数量有限。在这种情况下, f1-atpase 亚基被人工表达并组装成大肠杆菌复合物, 整个异源酶通过带标记的亚基通过亲和层析纯化. 这种方法导致测定了两种嗜热细菌的 f1-atpase 结构, 即 15 型硬毛虫和热性钙弧菌16 . 17. 然而, 这种方法相当不适合真核细胞 f-1-atpases, 因为它依赖于原核合成装置、翻译后处理和复杂的组装。

以氯仿为基础的提取剂以前被用来从单细胞二基因寄生虫trypanosoma cruzi 18和t. brucei19中分离出 f-1-atpase, 这是引起美国和美国的重要哺乳动物病原体.非洲锥虫酶, 分别来自单源昆虫寄生虫刺五加 20。这些净化只导致对 f1-atpase 的简单描述, 因为没有使用下游应用来充分描述该综合体的组成、结构和酶特性。本文介绍了一种从t.brucei 的培养昆虫生命周期阶段进行 f1-atpase 纯化的优化方法.该方法是在建立了牛和酵母 f1-atpase21、22分离方案的基础上发展起来的。该程序产生了高度纯、均匀的酶, 适用于体外酶和抑制检测、质谱仪23 的详细蛋白质组学表征和结构测定 24。纯化协议和原子级 f1-atpase 结构的知识为设计屏幕以识别小分子抑制剂提供了可能性, 并有助于开发抗非洲锥虫酶的新药.此外, 该协议还可用于从其他生物中净化 f 1-atpase。

Protocol

1. 缓冲器和解决方案 准备下面列出的解决方案。德加所有缓冲液的液相色谱。在使用前添加 adp、苯胺和蛋白酶抑制剂。 准备缓冲液 a:50 mm tris 缓冲液与盐酸 (tris-h科尔) ph 值 8.0, 0.25 m 蔗糖、5 mm 苯胺酸、5 mm 氨基己酸 (aca) 和蛋白酶抑制剂 (10μm 阿玛司他汀、50μm 酯蛋白、50μm 肽、50μm leupeptin 和50μm 二丙酸 a)。 准备缓冲液 b:50 mm Tris-HCl ph 值 8.0, 0.25 m 蔗糖, 4 mm 乙二胺四乙酸 (edt…

Representative Results

典型的纯化 (图 1) 从线粒体囊泡 (线粒体子体) 开始, 从低裂解的 1 x 1011到 2 x 10 11 顺环 t. 布鲁西细胞 25在标准培养中分离富葡萄糖 sdm-79 培养基 27.有丝体通过超声分裂, 旋转, 含有基质的上清液被丢弃。线粒体膜用氯仿处理, 释放 f1-atpase.离心后, 有机相和沉淀的中间相被丢弃。在强阴…

Discussion

在先前公布的从其他13、 14 种分离f1-atpase 复合物的方法基础上, 制定了 t. brucei 的f1-atpase 纯化方案.该方法不需要任何基因改造 (例如标记), 并产生一个完全活跃的复合体, 所有亚基都存在。关键的一步是氯仿促进的释放的 f1-atpase 从酶的膜连接部分.在对目前所描述的所有真核物种的纯化中, 释放的亚复合体在 1:3: 1:1:…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作由教育部 erc cz 赠款 ll久、捷克共和国赠款机构18-17529s 和 erdf/esf 寄生虫致病性和毒性研究项目中心资助 (否)。CZ.02.1.01/0.0/0.0/16_019/0000759)。

Materials

Chemicals
Adenosin Diphosphate Disodium Salt (ADP) Applichem A0948
Amastatin Hydrochloride Glantham Life Sciences GA1330
Aminocaproic Acid Applichem A2266
BCA Protein Assay Kit ThermoFischer Scientific/Pierce 23225
Benzamidine Hydrochloride Calbiochem 199001
Bestatin Hydrochloride Sigma Aldrich/Merck B8385
Chloroform Any supplier
cOmplete Tablets, Mini EDTA-free Roche 4693159001 Protease inhibitor cocktail tablets
Ethylenediaminetetraacetic Acid (EDTA) Any supplier
Hydrochloric Acid Any supplier For pH adjustment
Ile-Pro-Ile Sigma Aldrich/Merck I9759 Alias Diprotin A
Leupeptin Sigma Aldrich/Merck L2884
Magnesium Sulfate Heptahydrate Any supplier
Pepstatin A Sigma Aldrich/Merck P5318
Protein Electrophoresis System Any supplier
Sodium Chloride Any supplier
Sucrose Any supplier
Tris Any supplier
Name Company Catalog Number Comments
Consumables
Centrifuge Tubes for SW60Ti, Polyallomer Beckman Coulture 328874
DounceTissues Homogenizer 2 mL Any supplier
Glass Vacuum Filtration Device Sartorius 516-7017 Degasing solutions for liquid chromatography
HiTrap Q HP, 5 mL GE Healthcare Life Sciences 17115401 Anion exchange chromatography column
Regenaretad Cellulose Membrane Filters, pore size 0.45 μm, diameter 47 mm Sartorius 18406–47——N Degasing solutions for liquid chromatography
Superdex 200 Increase 10/300 GL GE Healthcare Life Sciences 29091596 Size-exclusion chromatography column
Vivaspin 6 MWCO 100 kDa PES Sartorius VS0641
Name Company Catalog Number Comments
Equipment
AKTA Pure 25 GE Healthcare Life Sciences 29018224 Or similar FPLC system
Spectrophotometer Shimadzu UV-1601 Shimadzu Or similar spectrophotometer with kinetic assay mode
Ultracentrifuge Beckman Optima with SW60Ti Rotor Beckman Coulture Or similar ultracentrifuge and rotor
Ultrasonic Homogenizer with Thin Probe, Model 3000 BioLogics 0-127-0001 Or similar ultrasonic homogenizer
DOWNLOAD MATERIALS LIST

References

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Tags

F1-ATPaseTrypanosoma BruceiMitoplastsProtein PurificationMitochondrial MembraneSonicationUltracentrifugationChloroform ExtractionDounce Homogenizer

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Gahura, O., Zíková, A. Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei. J. Vis. Exp. (143), e58334, doi:10.3791/58334 (2019).
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