The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).

1.执行与纯蛋白水解ATP反应<ol><li>备齐试剂的股票与纯蛋白孵化。 <ol><li>制备含有100mM HEPES pH 8.5的65毫摩尔NaCl和5％甘油（或其它测定缓冲液适当）5倍的HEPES /氯化钠/甘油（HNG）缓冲器。 </li><li>制备的100mM的MgCl 2（或其他金属，如果ATP酶是金属依赖性）的水。 </li><li>准备新鲜的100毫米ATP在200毫米的Tris基地使用高纯度的ATP（不进一步调整pH值）。等分试样并储存的ATP库存在-20℃下不超过几个星期长，作为ATP的会分解随着时间的推移，从冷冻和解冻的ATP库存避免。 </li><li>预混物的MgCl 2和ATP在1:1的比例只是之前设置ATP的水解反应。 </li></ol></li><li>准备和标记1.5毫升管的定期整个反应过程中采集样品。准备在试管时间0和时间15，30收集样品，45和60分钟。 注意:作为替代，只在时间0和端点收集样品。 <ol><li>添加245微升1×HNG缓冲向每个管中以稀释来自ATP的水解反应1时50收集的样品。 </li></ol></li><li>制备干冰和乙醇的浴快速冷冻样品，使反应停止。在橡胶冰桶或其他安全的（非塑料）容器中，加几片干冰和精心浇足70-100％的乙醇覆盖干冰。 </li><li>稀释纯化的蛋白质在1X HNG缓冲，适当（一般为5-10μM），并保持在冰上。 </li><li>成立了ATP水解反应。 <ol><li>在单独0.5毫升管每个样品中，添加以下试剂（按顺序）:H 2 O（最多30微升的最终体积），6微升的5倍的HNG或其他缓冲液，3微升100mM的MgCl 2 -ATP混合物和0.25-5μM蛋白质。 </li><li>包括一个缓冲器只阴性对照，其中不加入蛋白的条件。</li></ol></li><li>在时间0除去从反应5微升，在含有245微升的HNG缓冲液中制备的1.5毫升管稀释1:50，并立即冷冻在干冰/乙醇浴中的样品。 </li><li>孵育在37℃下反应以允许发生1小时ATP水解。在每个时间间隔（15，30，45，和60分钟），从反应除去5微升等份，并加入到含有HNG缓冲如在步骤1.6标记的样品管中。 </li><li>在ATP水解反应结束时，将稀释的样品到-80℃冷冻贮存。为了确保所有的样品完全冷冻，等待出发前至少10分钟。 </li></ol> 2.样品孵育含有游离皮与检测试剂<ol><li>在室温（从步骤1.6和1.7获得）包含在每个时间点ATP水解反应等分试样解冻稀释的样品。 </li><li>建立含有样品和磷酸盐标准的96孔板中。 <ol><li>在0.5ml的涂是，加入5.5微升800微米标准104.5微升HNG缓冲液稀释800微米至40微米的磷酸盐标准（带有皮检测试剂）。拌匀，并加入100微升此40μM丕标准，做好对A1 96孔板。 </li><li>加入50μlHNG缓冲井B1-H1为1:郫县标准的1系列稀释。 </li><li>从A1孔中取出50微升40μM丕并加入50微升分析缓冲液中以及B1，混合，并从B1孔取出50微升，并通过精心G1增加以及C1，继续稀释。混合，以保证每口井都有相同的体积后丢弃以及G1 50微升。那么H1应该只包含创建丕标准从40到0μM缓冲区。 注意:如果一个附加的因子（例如抑制剂）已被添加到样品中，创建包含该因子以控制在这些条件下在磷酸盐释放或吸光度的变化的标准曲线。 </li><li>加入50微升每个SAMP的勒式两份的板。和不同的时间点水平;从在列同一时间点垂直（样品2时间0 = B2，B3样品1时间0 = A2，A3）添加样品。这允许多达8个样品，每个板5的时间点。 </li></ol></li><li>使用无菌移液管，以除去足够的孔雀石绿/钼酸丕检测试剂至100μl添加至各含有样品和标准孔（试剂需要（毫升）=样品和标准的0.1×数），并加入到便于吸移一个菜使用多通道移液管。不直接倒入检测试剂放入盘中，因为皮污染可能发生。 </li><li>使用多通道移液管，从最后的时间点添加100微升丕检测试剂至各孔并混合通过小心地上下吹打次数一致的号码而不引入气泡，优选以所述第一时间点。 </li><li>孵育在室温下25分钟的盘，或根据马nufacturer的指示。 </li></ol> 3.定量结果使用酶标仪<ol><li>使用吸光度酶标仪读取样品的吸光度在650nm处。 </li><li>做一个皮标准曲线。使用绘图软件，图形，以便找到用于解决磷酸盐的每个样品中的量的方程为PI标准样品与浓度的吸光度值。 </li><li>通过用磷酸盐标准校准计算各样品的ATP酶活性。磷酸盐释放=（OD 650 - Y轴截距）/斜率。 <ol><li>从每个样品的重复平均总皮。减去这个数字的唯一缓冲控制的吸光度。稀释倍数乘以（在本例中50）。 </li><li>确定每个微摩尔蛋白释放纳摩尔皮。在动力学分析作图对于每个时间点，这些值应产生至少R = 0.99（ 图1）的线性拟合;如果n催产素，该测定可以与更多或更少的蛋白质或长或短的孵育时间进行调整。 </li></ol></li><li>表示的结果以nmolπ/微摩尔蛋白/分钟（ 图2,3），或以nmolπ/微克蛋白/分钟如果需要的话。 </li></ol>

<ol>
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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=Decoupling+catalytic+activity+from+biological+function+of+the+ATPase+that+powers+lipopolysaccharide+transport.">Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.</a> Proc Natl Acad Sci U S A. 111 (13), 4982-4987 (2014).</li><li>Zhang, X., 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=Altered+cofactor+regulation+with+disease-associated+p97/VCP+mutations.">Altered cofactor regulation with disease-associated p97/VCP mutations.</a> Proc Natl Acad Sci U S A. 112 (14), E1705-E1714 (2015).</li><li>Rowlands, M. G., Newbatt, Y. M., Prodromou, C., Pearl, L. 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The authors have nothing to disclose.

这是用于对生化表征纯化的蛋白质的体外 ATP酶活性测定通用协议。该方法很容易进行优化;例如，调整的蛋白质，缓冲液和盐的组合物，温度，以及改变该测定长度和间隔（包括增加的间隔的总数）的量可提高活性定量。市售孔雀石基于绿色试剂都高度敏感，而且可以检测少量游离磷酸的（〜100微升50皮摩尔）。因为该测定的敏感性，这是至关重要的使用一次性塑料器皿，超纯水，缓...

ATPases are integral enzymes in many processes across all kingdoms of life. ATPases act as molecular motors that use the energy of ATP hydrolysis to power such diverse reactions as protein trafficking, unfolding, and assembly; replication and transcription; cellular metabolism; muscle movement; cell motility; and ion pumping1-3. Some ATPases are transmembrane proteins involved in transporting solutes across membranes, others are cytoplasmic and may be associated with a biological membrane such as the plasma membrane or those of organelles.
AAA+ ATPases (ATPases associated with various cellular activities) make up a large group of ATPases that share some sequence and structural conservation. These proteins contain conserved nucleotide binding motifs such as Walker-A and -B boxes and form oligomers (generally hexamers) in their active state1. Large conformational changes in these proteins upon nucleotide binding have been characterized among diverse members of the AAA+ family. EpsE is a AAA+ ATPase and member of the bacterial Type II/IV secretion subfamily of NTPases4-6. EpsE powers Type II Secretion (T2S) in Vibrio cholerae, the causative agent of cholera. The T2S system is responsible for the secretion of a wide variety of proteins, such as the virulence factor cholera toxin that causes profuse watery diarrhea when V. cholerae colonizes the human small intestine7. 
Techniques for quantitating in vitro ATPase activity are varied, but commonly measure phosphate release using colorimetric, fluorescent, or radioactive substrates8-11. We describe a basic method for determining in vitro ATPase activity of purified proteins using a colorimetric assay based on a commercially available malachite green-containing substrate that measures liberated inorganic phosphate (Pi). At low pH, malachite green molybdate forms a complex with Pi and the level of complex formation can be measured at 650 nm. This simple and sensitive assay may be used to functionally characterize new ATPases and to evaluate the roles of potential activators or inhibitors, to determine the importance of domains and/or specific residues, or to assess the effect of particular treatments on enzymatic activity.

<table><tbody><tr><td>HEPES buffer</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358-212</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Fisher</td><td>BP41325</td><td></td></tr><tr><td>96-well plates (clear, flat-bottom)</td><td>VWR</td><td>82050-760</td><td></td></tr><tr><td>BIOMOL Green</td><td>Enzo Life Sciences</td><td>BML-AK111</td><td>Preferred phosphate detection reagent. Caution: irritant.</td></tr><tr><td>Microplate reader</td><td></td><td></td><td>BioTek Synergy or comparable</td></tr><tr><td>Prism 5</td><td>GraphPad Software</td><td></td><td></td></tr></tbody></table>

measuring in vitro atpase activity for enzymatic characterization

三磷酸腺苷水解酶，或ATP酶，在细胞功能多样化阵列发挥关键作用。这些动态蛋白可用于机械工作，如蛋白质运输和退化，溶质运移和细胞运动产生能量。此处所描述的协议是用于测量纯化ATP酶的体外活性功能表征一个基本化验。蛋白水解的ATP以导致的无机磷酸盐的释放反应，和磷酸盐的释放的量，然后用比色测定法进行定量。这个高度适应协议可以进行调整，以测量在动力学或终点测定ATP酶活性。代表性的协议是基于活性和EPSE要求这里提供时，AAA + ATP酶参与II型分泌在细菌霍乱弧菌 。测量活动所需的纯化的蛋白质的量，测定的长度和sa定时和数量mpling间隔，缓冲液和盐的组合物，温度，辅因子，兴奋剂（如果有的话） 等可以从这里描述的变化，并且因此一些优化可能是必要的。该协议为特征的ATP酶的基本框架，并可以快速进行，方便地根据需要进行调整。

所述T2S ATP酶EPSE 的体外活性可通过EPSE的共纯化与EPSL（EPSE-cytoEpsL）的胞质结构域和另外的酸性磷脂心磷脂12的刺激。另外，也可以通过比较野生型（WT）的活性，使用该测定的蛋白质的变异形式，以确定在ATP水解特定EPSE残基的作用。这里，替换在EPSE锌结合域中的两个赖氨酸残基的作用是通过纯化的WT EPSE-cytoEpsL的ATP酶活性的比较对EPSE K417AK419A-cytoEpsL变体测定?...

Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

测量<em&gt;体外</em&gt;的酶学性质ATP酶活性

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

measuring-in-vitro-atpase-activity-for-enzymatic-characterization

Research

JoVE Journal

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

18.0K 次观看.  University of Michigan. We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

视频: Measuring In Vitro ATPase Activity for Enzymatic Characterization

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

科研

生物化学

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

使用新激酶蛋白的强生化方法在分子水平上进行表征

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

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 ...

生物学

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

免疫与感染

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

生化分析的分析ATP依赖的染色质重塑酶活性的影响

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

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.

体外试验来测量磷脂酰乙醇胺甲基转移酶活

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Source: Pokhrel, A., et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

基于薄层色谱的酶活性测定:一种使用薄层色谱法测定体外焦磷酸激酶活性的技术

Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
This ...

测量

摘要

探索更多视频

此视频中的章节

用放射性标记的ATP测定蛋白激酶活性

使用新激酶蛋白的强生化方法在分子水平上进行表征

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

醋酸形成醋酸酶激酶活性的直接检测

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的

生化分析的分析ATP依赖的染色质重塑酶活性的影响

体外试验来测量磷脂酰乙醇胺甲基转移酶活

基于薄层色谱的酶活性测定:一种使用薄层色谱法测定体外焦磷酸激酶活性的技术

测量

摘要

探索更多视频

此视频中的章节

用放射性标记的ATP测定蛋白激酶活性

使用新激酶蛋白的强生化方法在分子水平上进行表征

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

醋酸形成醋酸酶激酶活性的直接检测

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

生化分析的分析ATP依赖的染色质重塑酶活性的影响

体外试验来测量磷脂酰乙醇胺甲基转移酶活

基于薄层色谱的酶活性测定:一种使用薄层色谱法测定体外焦磷酸激酶活性的技术

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的