作者是帕金森的英国研究员（补助金F1002）。这项工作是支持部分由威康信托/湄公河委员会联合呼叫在神经退行性疾病奖（WT089698）的英国帕金森的疾病联盟（UKPDC）的神经学伦敦大学学院研究所，在谢菲尔德大学和在湄公河蛋白磷酸化组在邓迪大学。

安全 下面所描述的协议利用放射性32磷 ATP标记的γ磷酸遵循的LRRK2的激酶活性。它是基于在我们的实验室中使用的标准协议，并在如运行凝胶过程中的许多步骤， 免疫印迹等等的材料和精密的 ​​条件，应作为设备和协议的指导用于这些过程的变化，从实验室到实验室。含有同位素发射电离辐射可能危害人体健康和严格的许可证制度和法规的机构和国家一级控制其使用。在本议定书进行了实验在开源使用辐射伦敦大学学院的培训，并在学院提供的安全服务的指导方针的良好实验室操作规范（指引AVAilable <a href="http://www.ucl.ac.uk/estates/safetynet/training/glp_hurs.pdf">http://www.ucl.ac.uk/estates/safetynet/training/glp_hurs.pdf</a> ）。适当的培训和监管部门的批准之前，不应尝试使用开源的辐射。监管机构，负责开放源码辐射实验室研究因国家而异。这方面的例子是：在英国的健康与安全执行局（ <a href="http://www.hse.gov.uk/radiation/ionising/index.htm">http://www.hse.gov.uk/radiation/ionising/index.htm</a> ）在美国，核管理委员会（ <a href="http://www.nrc.gov/materials/miau/regs-guides-comm.html">http:// www.nrc.gov /材料/苗丰强/ REGS指南- comm.html</a> ），加拿大核安全委员会（ <a href="http://nuclearsafety.gc.ca/eng/">http://nuclearsafety.gc.ca/eng/</a> ），在加拿大和德国DAS Bundesamt献给Strahlenschutz（ <a href="http://www.bfs.de/de/bfs">HTTP ：/ / www.bfs.de / DE / BFS</a> ）。在其他国家的用户，应确认与辐射安全主任的地方性法规，规章和发牌当局。人们已经注意到，在文本本议定书有关的安全防范措施，突出具有放射性的三叶符号（ <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" /> ）。 1。准备激酶反应<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />所有反应混合物的准备1.5毫升样品管中含有一个O形环，以防止放射性扩散的螺丝帽。 <ol><li>在冰上解冻蛋白 - 野生型，D1994A，LRRK2 G2019S。 </li><li>弥补冰 - 0.5μg/μlMBP的LRRK2，10NM，5ul的10X激酶缓冲，使水以50μL的反应。 </li></ol> 2。运行检测<img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />利用32个P ATP的所有步骤应取p花边在指定的辐射区域。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />合适的个人防护装备应戴 - 根据本实验室的标准作业程序，其中包括实验室外套，双层手套和护目镜。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />样品含有32个P ATP应该屏蔽6毫米的有机玻璃屏幕，以尽量减少暴露用户。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />在适用情况下，个人监测设备应使用 - 在伦敦大学学院，任何认证的开放源码的辐射用户必须有一个电影的徽章监控在实验过程中的辐射暴露。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />应该评估所有实验表面放射性污染之前和之后使用使用的基GER计数器。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />所有可能被污染的消耗品应处置放射性废物处置的机构准则的严格遵守。 <ol><li>开始检测之前，到30 ° C和100 ° C分别设置加热块</li><li>取出-20冰箱（请注意，储存条件，从32个P ATP可能会有所不同供应商或使用的radionucleotides 类型而定 ）的32个P ATP。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />扫描以外的容器，使用前，有机玻璃屏幕背后解冻。 </li><li>在冰上的反应， 新增32个 P ATP加入1μl，随着冷ATP 10微米。 </li><li>用吸管拌匀。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />脉冲离心机把液体的试管底部，减少污染的危险。 </li><li>零时间点取出15μl等份ð终止此外，5μL4X SDS样品缓冲液和变性反应在分装在100 ° C时10分钟。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />脉冲离心机把液体的试管底部，减少污染的危险。 </li><li>剩余样品放置在加热块，并在30 ° C孵育60分钟。 </li><li> 15μl删除10分钟，60分钟的时间点，并在100此外4X SDS样品缓冲液和变性5μL终止反应° C。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />脉冲离心机把液体的试管底部，减少污染的危险。 </li></ol> 3。免疫样品和分析结果<ol><li>样本上运行的SDS - PAGE <ol><li> 10 4-12％二 - 三polyacrilamide凝胶电泳准备使用MOPS运行缓冲。 </li><li>每个样品20μL装上凝胶锐器7μlTAIN蛋白质标准的阶梯。 </li><li>凝胶在160V运行90分钟，或直至染料前沿到达凝胶年底。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />所有液体接触放射性同位素应作为放射性废物处理和处置按机构指引。伦敦大学学院规定的状态，液体放射性废物的，应当由具有丰富的水倾泻而下指定的放射性处置汇丢弃。 </li></ol></li><li> 通过免疫印迹蛋白转移到PVDF膜上。 <ol><li>传输缓冲区准备，1X三甘氨酸加20％甲醇。 PVDF膜和滤纸切，以正确的凝胶大小和膜或在滤纸的情况下转移缓冲液预湿的情况下，冰川甲醇激活。膜应标有圆珠笔油墨，以便识别取向。 <img src="/files/ftp_upload/3495/3495symbol1.jpg" alt="符号1" />凝胶中删除从PLASTIC外壳和多余的丙烯酰胺和处置放射性废物。 </li><li>凝胶应形成一个三明治膜和滤纸，确保无气泡膜和凝胶之间存在，然后安排在与膜之间的凝胶和阳极的免疫印迹仪器。 </li><li>转让在25V进行16个小时。 </li></ol></li><li>蛋白转移到PVDF膜，应在室温下干燥。最后，应该是孤立的干燥膜醋酸纤维素片之间，接触到一个荧光屏或X光片，让放射性同位素标记蛋白检测。曝光时间可以从几个小时，超过一周取决于使用放射性同位素和酶的反应动力学的具体活动。 </li><li> Phosphoscreen扫描/电影处理。如果使用荧光屏，图像应保存为一个高分辨率的TIFF或位图文件，如果使用胶片，然后由此产生的TRANsparency应该使用的是台式扫描仪扫描，并保存为高分辨率的TIFF或位图文件。分析图像，然后可以在ImageJ，一个免费程序，在国家卫生部网站（ <a href="http://rsbweb.nih.gov/ij/">上</a>提供http://rsbweb.nih.gov/ij/ ）。 </li></ol> 4。代表性的成果图1显示了代表进行了一个试验使用作为一种通用的磷酸受体底物髓鞘碱性蛋白野生型，G2019S和D1994A LRRK2结果。 LRRK2自身磷酸化≈200kDa是可见的，与代表从20 - 40kDa的磷酸化的MBP可见多个频段。注意在D1994A（激酶死）车道的情况下的自身磷酸化，并增加磷酸化由于G2019S突变。注意还残留的MBP磷酸化激酶死巷。这可能是由于不完全消融D1994A突变的LRRK2激酶活性，或反映的存在Øf跟踪污染的反应激酶。 <img src="/files/ftp_upload/3495/3495fig1.jpg" alt="图1" /> 图1。

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没有利益冲突的声明。

本文介绍了一种用于化验的LRRK2的激酶活性，在体外系统的基本协议。在简洁的利益，这已不限于一小时的终点模板，使用一个通用的基板，但一般协议是适用于潜在的承印物范围，适合更复杂的分析研究LRRK2激酶活性的动力学。这突出表明利用体外系统研究，像这样的一种蛋白激酶行为的关键优势之一：酶和底物的浓度，因为有完整的控制，它可以生成动力学数据和计算的K m和V 最大值反应。对于更详细的动力学评价或公认的抑制剂的影响进行评估的，更高的吞吐量系统（如使用LRRKtide基板，自Invitrogen提供）的是一个优越的alternati已经在这里介绍的方法。 这一点很重要，但是，认识到还原模型在体外激酶分析系统提供的是一个工具来检查生物学激酶和其潜在基板之间的关系。像这样的检测数据应在与其他方法结合使用激酶如何在体外和行为在一个细胞的背景下，以获得全面的了解。例如， 在体外磷酸化的一个公认的基板可以通过质谱分析，以确定有针对性的突变（如转换的丝氨酸或苏氨酸磷酸受体残基丙氨酸，如无phosphorylatable残留），然后可以通过操纵研究功能可能phosphosites磷酸体外的后果。 在体外检测系统如在解释数据的一个主要考虑因素是eithe可能性R型I或II型错误（假阳性或假阴性）。不幸的是，该系统的还原性质作主既 - 在前者的情况下，有一个纯化假定在人为地接近，并在高浓度的底物和纯化激酶（蜂窝环境相比）可能会导致artefactual磷酸化事件。相反，作为一个复杂的一部分，许多激酶的功能内的细胞中，并要求一个给定的基材发生磷酸化的共同因素。如上所述，从一个假定的底物磷酸化的积极结果在体外检测系统使用，那么应该是在蜂窝系统测试验证结果和阴性结果应谨慎解释。 鉴于这种情况，这是关键，可能有正反两方面的控制，让您感兴趣的激酶活性的比较研究。仔细选择了积极控器升，有一个对你感兴趣的蛋白质知名的活动，与阴性对照，是不太可能的磷酸化公认的基板，是极其宝贵的。一个LRRK2候选人控制受体相互作用的激酶3，其中有一个密切相关的主序列激酶结构域的LRRK2，但有一个非常不同的总体域结构18。这是从Invitrogen公司的重组蛋白，并作为标准控制在我们的实验室使用。 还应当指出的是，市售的LRRK2形式缺乏的蛋白质N -末端和谷胱甘肽- S -转移，这应该被视为一个潜在的混杂因素考虑在进行这种蛋白激酶检测标签，因为它是不知道什么样的作用N -末端的LRRK2这种蛋白质发挥正常功能。例如，如果在这个协议中的部分是缺席的蛋白质N -末端的LRRK2是招聘的LRRK2激酶结构域的一个特定的基板关键，那么这将有一个观察磷酸化产生重大影响，在体外系统的基板上面描述。 即使这些前提条件，然而，帕金森氏研究LRRK2生物学的重要性，强调作为一个有价值的工具，在调查这种蛋白质的行为 ，在体外设置本议定书的效用。

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> LRRK2野生型</td><td> Invitrogen公司</td><td> PV4873 </td><td></td></tr><tr><td> LRRK2 G2019S </td><td> Invitrogen公司</td><td> PV4881 </td><td></td></tr><tr><td> LRRK2 D1994A </td><td> Invitrogen公司</td><td> PV6051 </td><td></td></tr><tr><td>激酶缓冲</td><td>细胞信号</td><td> 9802S </td><td></td></tr><tr><td> MBP </td><td>西格玛</td><td> M1891 </td><td></td></tr><tr><td> 32P克三磷酸腺苷</td><td> Perkin Elmer公司</td><td> BLU002X500UC </td><td> 500μCi，30Ci / mMole </td></tr><tr><td> 4X LDS样品缓冲液</td><td> Invitrogen公司</td><td> NP0007 </td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td>西格玛</td><td> M6250 </td><td></td></tr><tr><td>蛋白标准</td><td> Invitrogen公司</td><td> LC5800 </td><td></td></tr><tr><td> MOPS电泳缓冲液</td><td> Invitrogen </td><td> NP0001 </td><td></td></tr><tr><td>双 - 三丙烯酰胺凝胶</td><td> Invitrogen公司</td><td> NP0321 </td><td> 4-12％1毫米10well </td></tr><tr><td> PVDF膜</td><td> Millipore公司</td><td> IPVH00010 </td><td></td></tr><tr><td>传输缓冲区</td><td> Invitrogen公司</td><td> NP0006 </td><td></td></tr></tbody></table>表1。试剂蒸馏水和去离子水用于所有稀释步骤<table border="1"><tbody><tr><td> 设备类型 </td><td> 公司 </td><td> 评论 </td></tr><tr><td>盖革计数器</td><td>微型仪器</td><td></td></tr><tr><td> SDS - PAGE罐</td><td> Invitrogen公司</td><td></td></tr><tr><td>转让坦克</td><td> Invitrogen公司</td><td></td></tr><tr><td>热块（2） </td><td> Eppendorf公司</td><td></td></tr><tr><td>荧光屏</td><td>通用电气公司</td><td> X光片可以代替</td></tr><tr><td>荧光粉我mager </td><td>通用电气公司</td><td></td></tr><tr><td>曝光卡带</td><td>通用电气公司</td><td></td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td></td></tr><tr><td>有机玻璃屏蔽</td><td></td><td></td></tr><tr><td> 1.5毫升管</td><td> VWR </td><td>与O形环的螺丝顶部</td></tr></tbody></table>表2。设备

assaying the kinase activity of lrrk2 in vitro

富亮氨酸重 ​​复激酶2（LRRK2）是一个2527氨基酸的蛋白质ROCO家族的成员，拥有一个复杂的，多域结构，包括一个GTP酶域（称为中华民国，复杂的蛋白质RAS）和一个激酶结构域1。 LRRK2突变LRRK2引起帕金森氏病（PD）在2004年发现导致体积庞大的研究重点，并到其正常功能的蛋白质如何去歪在疾病状态 2,3 。初步调查到的LRRK2的功能集中在 4-6酶活性。清晰的画面，虽然尚未出现在这些一致的改动由于突变，从一批批数据突出了LRRK2激酶活性在与突变7,8的细胞死亡的重要性。最近的出版物报道针对LRRK2激酶活性的抑制剂，提供了重要的实验工具9-11。鉴于这些数据，LRRK2的酶学性质，我们提供的这种蛋白质的生物学的一个重要窗口，虽然无论是帕金森氏症的潜在的药物靶标是公开辩论。 一些不同的方法已被用于检测激酶活性的LRRK2。最初，检测进行使用表位标记的蛋白在哺乳动物细胞中过度表达，免疫沉淀，进行检测，使用这种蛋白质固定在琼脂糖珠 4,5,7 。随后，纯化的重组解决方案LRRK2片段也被用来例如，GST标签的片段纯化昆虫细胞中含有残留LRRK2 12 970 2527。近日，丹尼尔斯等人的隔离解决方案，从人类胚胎肾细胞全长LRRK2，但这种蛋白质是没有广泛使用的13个。相比之下，GST融合截断LRRK2形式是商业availablE（Invitrogen公司，详见表1），并提供了一​​个方便的工具表明LRRK2激酶活性检测。 LRRK2激酶活性的几种不同的输出报告。根据Moesin 558苏氨酸的磷酸化，被称为LRRKtide - - LRRK2髓鞘碱性蛋白（MBP）作为一种通用的激酶底物和一个人工基质磷酸化，磷酸化的自身磷酸化都被用来作为有一系列公认的生理底物包括α-突触核蛋白，Moesin和4 EBP的14-17。这些蛋白质作为LRRK2基板的状态仍不清楚，下文所述的协议，将重点放在作为一种通用的基板使用MBP的检测LRRK2激酶活性，针对潜在的承印物范围，并指出这一制度的效用。

Watch this Scientific Journal Video about Assaying the Kinase Activity of LRRK2 in vitro at JoVE.com

Assaying the Kinase Activity of LRRK2 in vitro

富亮氨酸重复激酶2是一个大型的多域激酶，基因突变，其中最常见的帕金森氏病的遗传原因。这种蛋白激酶活性分析已被证明是在了解这种蛋白质的生物学和功能障碍的重要工具。在本文中，<em&gt;在体外</em&gt;化验的LRRK2激酶活性和描述及其突变体的选择，提供了一个实验系统研究假定基板和潜在的功能障碍疾病的LRRK2磷酸化。

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

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Research

JoVE Journal

Biology

Assaying the Kinase Activity of LRRK2 in vitro

22.4K 次观看.  UCL Institute of Neurology. 富亮氨酸重复激酶2是一个大型的多域激酶，基因突变，其中最常见的帕金森氏病的遗传原因。这种蛋白激酶活性分析已被证明是在了解这种蛋白质的生物学和功能障碍的重要工具。在本文中，在体外化验的LRRK2激酶活性和描述及其突变体的选择，提供了一个实验系统研究假定基板和潜在的功能障碍疾病的LRRK2磷酸化。

视频: Assaying the Kinase Activity of LRRK2 in vitro

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

科研

生物学

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

醋酸形成醋酸酶激酶活性的直接检测

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

测定蛋白酶体降解的植物无细胞系统

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.