The authors thank the Nikon Imaging Center at Istituto Italiano di Tecnologia, Genova, Italy (NIC@IIT).

<ol>
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The authors have nothing to disclose.

酶活性是在不同的层次，包括RNA转录，蛋白质合成，蛋白质易位，翻译后修饰，和蛋白质 - 蛋白质相互作用精细调节。通常，酶表达单独不占它的活性。 ABPP的开发是为了研究在其天然状态的蛋白质的活性。两个特征是必需的:共价地结合到感兴趣的酶和记者标签的活性位点的化学探针来检测探针标记的酶。 探针设计与合成的过程的关键点。探针必须具有对其靶足够亲和力和?...

常用的方法来研究的表达模式，交互和蛋白质的功能，包括用于鸟枪分析1,2，酵母液相色谱-质谱平台双杂交方法3,4和体外测定法，是有限的，因为它们是无法评估在其天然状态的蛋白质的活性。基于活动的蛋白谱（ABPP）可以用来填补这一空白。在这种方法中，小分子探针能够共价结合到所关注的酶的活性位点被偶联至一个报告基团，其允许目标检测。使用点击化学（CC），记者可以集成到探针或之后发生5,6-靶接合可以引入。后者程序需要使用含有适当的化学基团，探针如末端炔或叠氮化物，其可以与许多记者试剂经由SUC生物正交反应改性H作为铜（Ⅰ）催化胡伊斯根[3 + 2]环加成7-9或Staudinger连接10,11。 最近，我们公开的化合物ARN14686作为第一ABP为在体外和半胱氨酸水解酶，NAAA 12的体内检测。 NAAA催化饱和和单不饱和的FAE，包括油酰乙醇酰胺（OEA）和十六酰胺乙醇（PEA），这是抗炎核受体PPAR-α13-15的内源性激动剂的水解失活。 NAAA在巨噬细胞等单核细胞衍生的细胞主要表达，以及在B淋巴细胞14,16，这表明在先天免疫应答的调节作用。所述酶在粗面内质网中合成的非活性形式和通过自我蛋白酶机构17中的细胞的酸性区室被激活。该裂解自我蛋白酶生成新N端半胱氨酸（C131在小鼠和大鼠，C126在人中），即是亲核体负责FAE水解18,19。 NAAA活动的药理学抑制改变有利于现场应用工程师16,20,21增加细胞水平的FAE合成/降解的平衡。数β内酯和β内酰胺衍生物已显示出抑制NAAA活性高效力和选择性16,22-26。这些抑制剂作用通过催化半胱氨酸16,27,28号第酰化。 （ - [（S）-2-代氮杂环丁烷-3-基]氨基甲酸4- cyclohexylbutyl- N）16化合物ARN14686是基于全身活性，丝氨酸衍生的β内酰胺NAAA抑制剂的化学结构，ARN726设计。 ARN726的4-丁基环己基用C 9饱和脂族链轴承与叠氮化物载运记者标签随后CC缀合末端炔标签替换。我们选择了设计一个两步ABP到minimallý改变原始支架的结构，从而保持了探针NAAA的亲和力。此外，避免引入笨重标签，这样的探针可以是更适合于不是直接ABP 体内治疗。 ARN14686抑制NAAA具有高效力（hNAAA IC 50 = 6纳米，rNAAA IC 50 = 13纳米）通过形成与酶12的催化半胱氨酸的共价加合物。在活老鼠的实验表明，该探针是在捕捉NAAA在肺部表达选择性。采用高探针浓度（10μM 体外 ，10毫克/毫升静脉内，ⅳ）12时酸性神经酰胺，共享33-34％的同一性与NAAA另一个半胱氨酸酰胺酶，也被确定为一个低亲和力靶。我们也使用ARN14686研究活性NAAA在以下完全弗氏佐剂（CFA）29的施用发炎大鼠组织的存在。 在这里，我们列出了preparati协议上ARN14686（ 图1）并将其应用到NAAA激活体外的调查。作为一个例子，我们描述的实验程序来可视化NAAA的CFA给药后的大鼠爪子。在这个实验中，蛋白从爪组织探针静脉注射后萃取，ABP标记蛋白质进行与生物素叠氮化物以CC。生物素化的样品使用链霉珠富集，并执行蛋白质印迹。在另一应用中，我们描述了活性NAAA的荧光显微镜在小鼠肺从探针处理的小鼠的本地化。在这种情况下，将组织切片并部分经受CC为罗丹明加成。工作流方案， 如图2所示。

<table><tbody><tr><td>1,1&amp;rsquo;-sulfonyldiimidazole&amp;nbsp;</td><td>Sigma Aldrich</td><td>367818</td><td>Harmful</td></tr><tr><td>2-dipyridylcarbonate</td><td>Fluorochem</td><td>11331</td><td>Harmful</td></tr><tr><td>2-Methylbutan</td><td>Sigma Aldrich</td><td>M32631</td><td>Flamable, toxic,hazardous to the aquatic environment</td></tr><tr><td>4-(Dimethylamino)pyridine</td><td>Sigma Aldrich</td><td>107700</td><td>Toxic</td></tr><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>695092</td><td>Flammable, Corrosive</td></tr><tr><td>Acetonitrile&amp;nbsp;</td><td>Sigma Aldrich</td><td>34998</td><td>Flammable, Toxic</td></tr><tr><td>Activated charcoal</td><td>Sigma Aldrich</td><td>161551</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td>Harmful</td></tr><tr><td>Azide-PEG3-Biotin</td><td>Jena Biosciences</td><td>CLK-AZ104P4</td><td></td></tr><tr><td>Azide-PEG3-Fluor 545</td><td>Jena Biosciences</td><td>CLK-AZ109</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>Bio-spin columns</td><td>Biorad</td><td>732-6204</td><td></td></tr><tr><td>Biotin</td><td>Sigma Aldrich</td><td>B4501</td><td></td></tr><tr><td>Blocking buffer</td><td>Li-Cor Biosciences</td><td>927-40000</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250</td><td>Higly toxic</td></tr><tr><td>Bovin serum albumine (BSA)</td><td>Sigma Aldrich</td><td>A7030</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma Aldrich</td><td>B0126</td><td></td></tr><tr><td>Bruker Avance III 400</td><td>Bruker</td><td></td><td></td></tr><tr><td>Celite</td><td>Sigma Aldrich</td><td>419931</td><td>Health hazard</td></tr><tr><td>Ceric ammonium nitrate&amp;nbsp;</td><td>Sigma Aldrich</td><td>22249</td><td>Oxidizing, Harmful</td></tr><tr><td>Chloral hydrate</td><td>Sigma Aldrich</td><td>C8383</td><td>Higly toxic</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma Aldrich</td><td>209198</td><td>Toxic</td></tr><tr><td>Cyclohexadiene</td><td>Sigma Aldrich</td><td>125415</td><td>Flammable, Health hazard</td></tr><tr><td>Cyclohexane</td><td>Sigma Aldrich</td><td>34855</td><td>Flammable, Harmful, Health hazard, Environmental hazard</td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>34856</td><td>Harmful, Health hazard</td></tr><tr><td>Diethyl ether</td><td>Sigma Aldrich</td><td>296082</td><td>Flammable, Harmful</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Acros Organics</td><td>348441000</td><td></td></tr><tr><td>Dimethyl sulfoxide &lt;em&gt;d&lt;sub&gt;6&lt;/sub&gt;&lt;/em&gt; (DMSO-&lt;em&gt;d&lt;sub&gt;6&lt;/sub&gt;&lt;/em&gt;)</td><td>Sigma Aldrich</td><td>175943</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>2860</td><td>Flammable, Harmful</td></tr><tr><td>Ethyl acetate</td><td>Sigma Aldrich</td><td>34858</td><td>Flammable, Harmful</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td></td></tr><tr><td>Irdye 680-LT Streptavidin</td><td>Li-Cor Biosciences</td><td>925-68031</td><td></td></tr><tr><td>IRDye680-LT Streptavidin&amp;nbsp;</td><td>Licor</td><td>925-68031</td><td>Briefly centrifuge before use to precipitate protein complexes</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>34966</td><td>Highly toxic</td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>34860</td><td>Flammable, Toxic, Health hazard</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-Dimethylaminopropyl)-&lt;em&gt;N&amp;prime;&lt;/em&gt;-ethylcarbodiimide hydrochloride</td><td>Sigma Aldrich</td><td>E7750</td><td>Harmful, Corrosive</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-diisopropylethylamine</td><td>Sigma Aldrich</td><td>D125806</td><td>Flammable, Corrosive, Toxic</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethylformamide</td><td>Sigma Aldrich</td><td>227056</td><td>Flammable, Harmful, Health hazard</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Cbz-L-Serine</td><td>Fluorochem</td><td>M03053</td><td>Harmful</td></tr><tr><td>Nikon A1 confocal microscopy</td><td>Nikon</td><td></td><td>Read the user manual</td></tr><tr><td>NuPAGE 4-12% Bis-Tris gel</td><td>Thermo Fisher Scientific</td><td>NP0335BOX</td><td></td></tr><tr><td>Palladium on carbon</td><td>Sigma Aldrich</td><td>330108</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-anisidine</td><td>Sigma Aldrich</td><td>A88255</td><td>Toxic, Health hazard, Environmental hazard</td></tr><tr><td>Paraformaldehyde</td><td>sigma Aldrich</td><td>441244</td><td>Toxic, respiratory harmful, corrosive, falmable</td></tr><tr><td>Poly(ethylene glycol)&amp;nbsp;</td><td>Sigma Aldrich</td><td>P3265</td><td></td></tr><tr><td>ProLong Gold antifade mountant with DAPI&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>P36931</td><td>Avoid bubbles formation</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma Aldrich</td><td>P8340</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)&amp;nbsp;</td><td>Sigma Aldrich</td><td>L3771</td><td>Toxic, corrosive, falmmable</td></tr><tr><td>Sodium hydride&amp;nbsp;</td><td>Sigma Aldrich</td><td>452912</td><td>Flammable</td></tr><tr><td>Sodium sulfate</td><td>Sigma Aldrich</td><td>239313</td><td></td></tr><tr><td>Starion FLA-9000 immage scanner</td><td>FUJIFILM</td><td></td><td>Read&amp;nbsp; the user manual</td></tr><tr><td>Streptavidin agarose</td><td>Thermo Fisher Scientific</td><td>20349</td><td></td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td></td></tr><tr><td>Tert-butanol</td><td>Sigma Aldrich</td><td>360538</td><td>Toxic, flammable</td></tr><tr><td>Tetrahydrofuran</td><td>Sigma Aldrich</td><td>186562</td><td>Flammable, Harmful, Health hazard</td></tr><tr><td>Thiourea</td><td>Acros Organics</td><td>424542500</td><td>Toxic, warm at 50 &amp;deg;C to dissolve</td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>RDD008</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine (TCEP)</td><td>Sigma Aldrich</td><td>C4706</td><td></td></tr><tr><td>Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td><td>Sigma Aldrich</td><td>678937</td><td></td></tr><tr><td>Triton-x100</td><td>Sigma Aldrich</td><td>X100</td><td>Toxic</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P9416</td><td></td></tr><tr><td>Tween-80</td><td>Sigma Aldrich</td><td>P1754</td><td></td></tr><tr><td>Ultra turrax IKA T18 basic tissue homogenizer</td><td>IKA</td><td></td><td></td></tr><tr><td>Undec-10-yn-1-ol</td><td>Fluorochem</td><td>13739</td><td>Harmful</td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U5378</td><td>Toxic, warm at 50 &amp;deg;C to dissolve</td></tr></tbody></table>

preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase

基于活动的蛋白谱（ABPP）是用于通过使用靶向酶的活性位点的化学探针的识别在复杂蛋白质组感兴趣的酶的方法。导入探针A记者标记允许通过在凝胶荧光扫描，蛋白质印迹，荧光显微镜，或液相色谱 - 质谱法检测标记的酶。这里，我们描述了制备和使用所述化合物ARN14686的，基于活动点击化学探针（CC-ABP），其选择性识别酶Ñ-acylethanolamine酸酰胺酶（NAAA）。 NAAA是半胱氨酸水解酶，通过去激活内源性过氧化物酶体增殖物激活受体（PPAR）-α激动剂如十六酰胺乙醇（PEA）和油酰乙醇酰胺（OEA）促进炎症。 NAAA被合成为一个无效的全长酶原，由autoproteolysis在溶酶体的酸性pH激活。定位研究ħ表明NAAA在巨噬细胞等单核细胞衍生的细胞在B淋巴细胞中表达，以及AVE。我们提供的ARN14686如何能够被用来检测和通过蛋白质印迹和荧光显微镜量化活性NAAA 离体啮齿动物组织的例子。

ARN14686是基于NAAA抑制剂ARN726的支架设计。 ARN726的4-丁基环己基用C 9饱和脂族链带有终端炔标签（ 图1）被取代。炔标签是为了允许使用两步标记过程的添加荧光团或通过CC一个生物素分子引入。这一功能使得ARN14686一个非常通用的工具，以探测NAAA 在体外和体内 。 这里，我们显示ARN14686的两个应...

Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

这里，我们描述一个基于活动的探针的制备和使用（ARN14686，十一碳-10- ynyl- -N - [（3 S）-2-代氮杂环丁烷-3-基]氨基甲酸），允许的检测和定量促炎酶ñ-acylethanolamine酸酰胺酶的活性形式（NAAA）， 在体外和离体 。

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

preparation-vivo-use-an-activity-based-probe-for-n-acylethanolamine

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

9.6K 次观看.  Istituto Italiano di Tecnologia. 这里，我们描述一个基于活动的探针的制备和使用（ARN14686，十一碳-10- ynyl- -N - [（3 S）-2-代氮杂环丁烷-3-基]氨基甲酸），允许的检测和定量促炎酶ñ-acylethanolamine酸酰胺酶的活性形式（NAAA）， 在体外和离体 。 

视频: Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

固定化酶和稳定的疏水盐改性的Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

科研

生物工程

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

丝状真菌曲霉菌的单步富集蛋白脱乙酰化酶的酶活性测定

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

生物化学

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

神经科学

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

基于NMR的活性测定,用于测定核苷裂解酶的化合物抑制、IC50值、伪影活性和全细胞活性

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

化学

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

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5.
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using &delta;13C or ion fragments of these biomarkers in biogeochemical studies9, 10.

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

衍生Aldononitrile乙酸氨基葡萄糖和胞壁酸的微生物在土壤中残留量的测定气相色谱的检测

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, high costs, and low patient numbers. Hundreds of mutant alleles are implicated in most of the LSDs. The diseases are typically classified into 2 to 3 different clinical types according to severity. Moreover, molecular characterization of the genotype can help predict clinical outcomes and inform patient care. Therefore, we developed a simple cell culture assay based on HEK293H cells heterologously over-expressing the mutations identified in Fabry and Pompe disease. A similar assay has recently been introduced as a preclinical test to identify amenable mutations for Pharmacological Chaperone Therapy (PCT) in Fabry disease. This manuscript describes an amended cell culture assay which enables rapid phenotypic assessment of allelic variants in Fabry and Pompe disease to identify eligible patients for PCT and may aid in the development of novel pharmacochaperones.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

体外酶法检测药物陪护与庞贝氏症的反应

The use of personalized medicine to treat rare monogenic diseases like lysosomal storage disorders (LSDs) is challenged by complex clinical trial designs, ...

医学

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 ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

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