Name: isolation of f1-atpase from the parasitic protist trypanosoma brucei
Uploaded: 2019-01-22T14:00:00
Description: Watch this Scientific Journal Video about Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei at JoVE.com

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)。

1. 缓冲器和解决方案
<ol><li>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。<ol>
<li>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)。</li>
		<li>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...

<ol>
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Structure and function of submitochondrial particles completely resolved with respect to coupling factor.</a> The Journal of Biological Chemistry. 242 (10), 2547-2551 (1967).</li><li>Senior, A. E., Brooks, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mitochondrial+oligomycin-insensitive+ATPase.+I.+An+improved+method+of+purification+and+the+behavior+of+the+enzyme+in+solutions+of+various+depolymerizing+agents.">Studies on the mitochondrial oligomycin-insensitive ATPase. I. An improved method of purification and the behavior of the enzyme in solutions of various depolymerizing agents.</a> Archives of Biochemistry and Biophysics. 140 (1), 257-266 (1970).</li><li>Tzagoloff, A., Meagher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+the+mitochondrial+membrane+system.+V.+Properties+of+a+dispersed+preparation+of+the+rutamycin-sensitive+adenosine+triphosphatase+of+yeast+mitochondria.">Assembly of the mitochondrial membrane system. V. Properties of a dispersed preparation of the rutamycin-sensitive adenosine triphosphatase of yeast mitochondria.</a> The Journal of Biological Chemistry. 246 (23), 7328-7336 (1971).</li><li>Beechey, R. B., Hubbard, S. A., Linnett, P. E., Mitchell, A. D., Munn, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+the+preparation+of+adenosine+triphosphatase+from+submitochondrial+particles.">A simple and rapid method for the preparation of adenosine triphosphatase from submitochondrial particles.</a> Biochemical Journal. 148 (3), 533-537 (1975).</li><li>Tyler, D. D., Webb, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+the+adenosine+triphosphatase+released+from+the+liver+mitochondrial+membrane+by+chloroform.">Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform.</a> Biochemical Journal. 178 (2), 289-297 (1979).</li><li>Hack, E., Leaver, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alpha-subunit+of+the+maize+F1-ATPase+is+synthesised+in+the+mitochondrion.">The alpha-subunit of the maize F1-ATPase is synthesised in the mitochondrion.</a> The EMBO Journal. 2 (10), 1783-1789 (1983).</li><li>Dunn, P. P., Slabas, A. R., Moore, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+F1-ATPase+from+cuckoo-pint+(Arum+maculatum)+mitochondria.+A+comparison+of+subunit+composition+with+that+of+rat+liver+F1-ATPase.">Purification of F1-ATPase from cuckoo-pint (Arum maculatum) mitochondria. A comparison of subunit composition with that of rat liver F1-ATPase.</a> Biochemical Journal. 225 (3), 821-824 (1985).</li><li>Satre, M., Bof, M., Vignais, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+Escherichia+coli+adenosine+triphosphatase+with+aurovertin+and+citreoviridin:+inhibition+and+fluorescence+studies.">Interaction of Escherichia coli adenosine triphosphatase with aurovertin and citreoviridin: inhibition and fluorescence studies.</a> Journal of Bacteriology. 142 (3), 768-776 (1980).</li><li>Abrahams, J. P., Leslie, A. G., Lutter, R., Walker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+at+2.8+&#197;+resolution+of+F1ATPase+from+bovine+heart+mitochondria.">Structure at 2.8 &#197; resolution of F1ATPase from bovine heart mitochondria.</a> Nature. 370 (6491), 621-628 (1994).</li><li>Lutter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+F1-ATPase+from+bovine+heart+mitochondria.">Crystallization of F1-ATPase from bovine heart mitochondria.</a> Journal of Molecular Biology. 229 (3), 787-790 (1993).</li><li>Kabaleeswaran, V., Puri, N., Walker, J. E., Leslie, A. G., Mueller, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+features+of+the+rotary+catalytic+mechanism+revealed+in+the+structure+of+yeast+F1-ATPase.">Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1-ATPase.</a> The EMBO Journal. 25 (22), 5433-5442 (2006).</li><li>Shirakihara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+thermophilic+F1-ATPase+inhibited+by+an+epsilon-subunit:+deeper+insight+into+the+epsilon-inhibition+mechanism.">Structure of a thermophilic F1-ATPase inhibited by an epsilon-subunit: deeper insight into the epsilon-inhibition mechanism.</a> The FEBS Journal. 282 (15), 2895-2913 (2015).</li><li>Stocker, A., Keis, S., Cook, G. M., Dimroth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+crystallization,+and+properties+of+F1-ATPase+complexes+from+the+thermoalkaliphilic+Bacillus+sp.+strain+TA2.A1.">Purification, crystallization, and properties of F1-ATPase complexes from the thermoalkaliphilic Bacillus sp. strain TA2.A1.</a> Journal of Structural Biology. 152 (2), 140-145 (2005).</li><li>Ferguson, S. A., Cook, G. M., Montgomery, M. G., Leslie, A. G., Walker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+thermoalkaliphilic+F1-ATPase+from+Caldalkalibacillus+thermarum.">Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (39), 10860-10865 (2016).</li><li>Cataldi de Flombaum, M. A., Frasch, A. C. C., Stoppani, A. O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+triphosphatase+from+Trypanosoma+cruzi:+purification+and+properties.">Adenosine triphosphatase from Trypanosoma cruzi: purification and properties.</a> Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 65 (1), 103-109 (1980).</li><li>Williams, N., Frank, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+ATP+synthase+of+Trypanosoma+brucei:+isolation+and+characterization+of+the+intact+F1+moiety.">The mitochondrial ATP synthase of Trypanosoma brucei: isolation and characterization of the intact F1 moiety.</a> Molecular and Biochemical Parasitology. 43 (1), 125-132 (1990).</li><li>Higa, A. I., Cazzulo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mg2+-activated+adenosine+triphosphatase+from+Crithidia+fasciculata:+purification+and+inhibition+by+suramin+and+efrapeptin.">Mg2+-activated adenosine triphosphatase from Crithidia fasciculata: purification and inhibition by suramin and efrapeptin.</a> Molecular and Biochemical Parasitology. 3 (6), 357-367 (1981).</li><li>Walker, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+and+subunit+stoichiometry+of+F1-ATPase+from+bovine+mitochondria.">Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.</a> Journal of Molecular Biology. 184 (4), 677-701 (1985).</li><li>Mueller, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ni-chelate-affinity+purification+and+crystallization+of+the+yeast+mitochondrial+F1-ATPase.">Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.</a> Protein Expression and Purification. 37 (2), 479-485 (2004).</li><li>Gahura, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+F1-ATPase+from+Trypanosoma+brucei+is+elaborated+by+three+copies+of+an+additional+p18-subunit.">The F1-ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.</a> The FEBS Journal. 285 (3), 614-628 (2018).</li><li>Montgomery, M. G., Gahura, O., Leslie, A. G. W., Zikova, A., Walker, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+synthase+from+Trypanosoma+brucei+has+an+elaborated+canonical+F1-domain+and+conventional+catalytic+sites.">ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (9), 2102-2107 (2018).</li><li>Schneider, A., Charriere, F., Pusnik, M., Horn, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+from+procyclic+Trypanosoma+brucei.">Isolation of mitochondria from procyclic Trypanosoma brucei.</a> Methods in Molecular Biology. 372, 67-80 (2007).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Wirtz, E., Leal, S., Ochatt, C., Cross, G. 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M., Nebohacova, M., Simpson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+I-complex+in+Leishmania+tarentolae+is+an+uniquely-structured+F1-ATPase.">The I-complex in Leishmania tarentolae is an uniquely-structured F1-ATPase.</a> Molecular and Biochemical Parasitology. 135 (2), 221-224 (2004).</li><li>Carbajo, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+N-terminal+domain+of+the+OSCP+subunit+of+bovine+F1Fo-ATP+synthase+interacts+with+the+N-terminal+region+of+an+alpha+subunit.">How the N-terminal domain of the OSCP subunit of bovine F1Fo-ATP synthase interacts with the N-terminal region of an alpha subunit.</a> Journal of Molecular Biology. 368 (2), 310-318 (2007).</li><li>Bowler, M. W., Montgomery, M. G., Leslie, A. G., Walker, J. 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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:...

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。

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosin Diphosphate Disodium Salt (ADP)</td>
			<td>Applichem</td>
			<td>A0948</td>
			<td></td>
		</tr>
		<tr>
			<td>Amastatin Hydrochloride</td>
			<td>Glantham Life Sciences</td>
			<td>GA1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminocaproic Acid</td>
			<td>Applichem</td>
			<td>A2266</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>ThermoFischer Scientific/Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzamidine Hydrochloride</td>
			<td>Calbiochem</td>
			<td>199001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bestatin Hydrochloride</td>
			<td>Sigma Aldrich/Merck</td>
			<td>B8385</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Tablets, Mini EDTA-free</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>Protease inhibitor cocktail tablets</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic Acid (EDTA)</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Any supplier</td>
			<td></td>
			<td>For pH adjustment</td>
		</tr>
		<tr>
			<td>Ile-Pro-Ile</td>
			<td>Sigma Aldrich/Merck</td>
			<td>I9759</td>
			<td>Alias Diprotin A</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Sigma Aldrich/Merck</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate Heptahydrate</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Sigma Aldrich/Merck</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Electrophoresis System</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes for SW60Ti, Polyallomer</td>
			<td>Beckman Coulture</td>
			<td>328874</td>
			<td></td>
		</tr>
		<tr>
			<td>DounceTissues Homogenizer 2 mL</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Vacuum Filtration Device</td>
			<td>Sartorius</td>
			<td>516-7017</td>
			<td>Degasing solutions for liquid chromatography</td>
		</tr>
		<tr>
			<td>HiTrap Q HP, 5 mL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17115401</td>
			<td>Anion exchange chromatography column</td>
		</tr>
		<tr>
			<td>Regenaretad Cellulose Membrane Filters, pore size 0.45 &#956;m, diameter 47 mm</td>
			<td>Sartorius</td>
			<td>18406--47------N</td>
			<td>Degasing solutions for liquid chromatography</td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29091596</td>
			<td>Size-exclusion chromatography column</td>
		</tr>
		<tr>
			<td>Vivaspin 6 MWCO 100 kDa PES</td>
			<td>Sartorius</td>
			<td>VS0641</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA Pure 25</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29018224</td>
			<td>Or similar FPLC system</td>
		</tr>
		<tr>
			<td>Spectrophotometer Shimadzu UV-1601</td>
			<td>Shimadzu</td>
			<td></td>
			<td>Or similar spectrophotometer with kinetic assay mode</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Beckman Optima with SW60Ti Rotor</td>
			<td>Beckman Coulture</td>
			<td></td>
			<td>Or similar ultracentrifuge and rotor</td>
		</tr>
		<tr>
			<td>Ultrasonic Homogenizer with Thin Probe, Model 3000</td>
			<td>BioLogics</td>
			<td>0-127-0001</td>
			<td>Or similar ultrasonic homogenizer</td>
		</tr>
	</tbody>
</table>

isolation of f1-atpase from the parasitic protist trypanosoma brucei

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.

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...

Watch this Scientific Journal Video about Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei at JoVE.com

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

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。

寄生性锥虫锥虫锥虫的 f1-atpase 的分离

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

F1-ATPase 的纯化基于一种经典的生化方法，这证明对于我们了解其他 ATP 合成器的 ATP 生产机制至关重要。该技术的主要优点是，它产生一种高度纯净的酶，适合通过X射线晶体学或低温电子显微镜进行结构测定。虽然此协议针对 F1-ATPase 与锥形体进行分离进行了优化，但它可以适应其他来源，如组织或培养细胞，以及各种致病性蛋白酶。这项技术可能导致新药的识别，以治疗严重的人类疾病。例如，结核分枝杆菌F-ATPase是结核病治疗的经过认证的药物靶点。为了开始协议，解冻并轻轻地重新暂停线粒体，也被称为米托皮斯特，以前通过低吨位解解从1倍10到11到2倍10到11个细胞的青霉蛋白锥虫布鲁西在5毫升的冰冷的缓冲剂 A.保持样品冷却。接下来，根据制造商的说明，确定悬浮液中的蛋白质浓度，即BCA蛋白测定。在超纯水中执行 BSA 稀释系列，以构建标准曲线。用超纯水稀释少量样品 20 至 100 次，以符合 BSA 标准的范围。计算样本中的总蛋白质量后，通过额外的缓冲器 A 稀释蛋白质浓度，将蛋白质浓度控制在每毫升 16 毫克，然后用声波将米托普斯特片切成倒置囊泡和膜片，每次声波 15 秒，总能量为 70 至 100 焦耳/脉冲，微尖直径为 3.9 毫米。在破碎时，在脉冲之间孵育样品在冰上30秒。声波化后，悬浮液可能会显得稍微暗一些。在摄氏4度下，以5.4万克的超离心沉淀膜碎片，在4摄氏度下以9.8万克沉积5小时。去干上一样物，然后进行氯仿萃取。根据使用的缓冲区 A 的总量计算缓冲区 B 的体积。将冷冻沉积物从离心管转移到均质器中。借助小型 Dounce 均质器，在缓冲器 B 中重新吸收线粒体膜的颗粒。将悬浮液转移到 50 毫升锥形管中。从冰中去除样品，将样品和所有溶液保持室温，以执行其余步骤。然后加入饱和的氯仿，用HCl调整为pH 8.5，一对一。紧闭盖子，用力摇动样品整整20秒。立即将混合物在8，400克下离心，在室温下5分钟。接下来，将上部、多云水相转移到1.6毫升微管。在样品中加入蛋白酶抑制剂，以取代氯仿处理去除的抑制剂。在室温下以13，000克离心样品30分钟。旋转后，将上经剂转移到新鲜的微管中，然后重复离心以去除任何剩余的不溶性材料。平衡连接到快速蛋白液相色谱系统的五毫升阴离子交换Q柱，其Q柱缓冲液流量为每分钟5毫升，直到吸收率在280纳米且电导率稳定。以每分钟一毫升的流速将上流器加载到平衡柱上。等待，直到 280 纳米的吸收度稳定在背景。以每分钟 0.5 毫升的流速，将 Q 柱洗脱缓冲液的 25 毫升线性梯度从 0%到 100%应用，并收集 1 毫升分数。在pH 8.0的pH8.0下，铂尔曼ATP酶测定，每分微升与ATP水解活性的主要洗脱峰值对应10微升。汇集显示 ATPase 活动的分数。使用具有 100，000 分子量截止 PES 过滤器的旋转柱，通过膜超滤将池样本浓缩到 200 到 500 微升。然后进行大小排除色谱。以每分钟 0.5 毫升的流速平衡连接到液相色谱系统的 Superose 6 增加柱，其至少 48 毫升的 SEC 缓冲液。将示例应用于列。以每分钟 0.25 毫升的流速运行色谱，同时收集 0.25 毫升分数。接下来，运行与 SDS-PAGE 上 UV 280 纳米吸收度轨迹峰值对应的 10 微升分数。然后用库马西蓝弄脏样品。通过铂尔曼 ATPase 测定，检测与包含 ATP 水解活性的 F1 峰值的第一个主要峰值对应的分数和 azide 灵敏度。然后进行 BCA 测定以确定蛋白质浓度。最后，将纯化的 F1-ATPase 保持室温，并在净化后三天内用于下游应用。这种黄离子交换色谱的洗脱轮廓显示洗脱缓冲液中 280 纳米的紫外线吸收度和氯化钠浓度。选定的分数在SDS-PAGE凝胶上分离，并沾染了库马西蓝色染料。对应于 Anion 交换色谱中主要洗脱峰值并包含 F1-ATPase 的分数被汇集、浓缩，并用作大小排除色谱的输入。主要污染物是二氢二苯二基脱氢酶，从大小排除色谱柱中脱氢作为离散峰。F1-ATPase 在第一个占主导地位的，主要是对称的峰中。通过 SDS-PAGE 分离纯化的 F1-ATPase 后，典型波段模式的 Coomassie 蓝色染色显示贝塔子单位上方可见的零星弱带，这些弱带表示 alpha 三个 beta 三头饰、200 度和 β 亚单位的寡聚体，并且没有质谱仪可检测到的任何污染物。在执行氯仿萃取（协议的关键步骤）时，必须大力摇动样品，并立即进行有机和水相的离心分离。按照此过程，通过质谱法可以详细描述隔离的 F1-ATPase。此外，它可用于活性测定或结构研究，无论是自己或存在各种抑制剂或基板类比。

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Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

Unit 1

Membrane Transport and Active Transporters

SCIENTISTS IN ACTION

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

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科研

生物化学

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

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Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

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Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

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Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

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Rapid Isolation of the Mitoribosome from HEK Cells

HEK 细胞 Mitoribosome 的快速分离

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Isolation of Tissue Extracellular Vesicles from the Liver

组织细胞外囊泡与肝脏分离

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Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

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Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

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Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

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