| A mant-GDP Dissociation Assay to Compare the Guanine Nucleotide Binding Preference of Small GTPases
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Author:
Date:
2021-01-20
[Abstract] Small GTPases are cellular switches that are switched on when bound to GTP and switched off when bound to GDP. Different small GTPase proteins or those with mutations may bind to GTP or GDP with different relative affinities. However, small GTPases generally have very high affinities for guanine nucleotides, rendering it difficult to compare the relative binding affinities for GTP and GDP. Here we developed a method for comparing the relative binding strength of a protein to GTP and GDP using a mant-GDP dissociation assay, whereby the abilities of GTP and GDP to induce the dissociation of bound mant-GDP are compared. This equilibrium type assay is simple, economic, and much faster than obtaining each protein’s affinity for GDP and GTP. The GDP/GTP preference value obtained is useful ...
[摘要] [摘要]小型GTPases是蜂窝交换机，绑定到GTP时打开，绑定到GDP时关闭。不同的小GTPase蛋白或具有突变的蛋白可能以不同的亲和力与GTP或GDP结合。然而，小的GTP酶通常具有非常高的鸟嘌呤核苷酸亲和力，使得难以比较GTP和GDP的相对结合亲和力。在这里，我们开发了一种方法，使用以下方法比较蛋白质与GTP和GDP的相对结合强度 mant-GDP解离分析，比较了GTP和GDP诱导绑定的mant-GDP解离的能力。这种平衡类型测定简单，经济并且比获得每种蛋白质对GDP和GTP的亲和力要快得多。Ť他GDP / GTP偏好值获得是用于比较的相对GTP有用/ GDP结合偏好小号不同GTP酶或不同的突变体，尽管这不是真正的GDP / GTP亲和力比（而是比的估计）。
[背景]小GTP酶（也称为小G蛋白）是一组功能上重要的大分子，由于其固有的水解酶活性，它们可以与GTP结合并水解为GDP 。在人类，米矿比一百种不同的小GTP酶分为五个家庭，即RAS，卢，冉，拉布和Arf的，根据其序列，结构，和功能（Wennerberg等，2005） 。
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| Preparation and Analysis of Crude Autolytic Enzyme Extracts from Staphylococcus aureus
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Author:
Date:
2015-12-20
[Abstract] The metabolism of the cell surface during bacterial cell division involves synthesis and degradation of peptidoglycan (PGN), the major component of the bacterial cell wall. Bacteria have to ensure that their surface remains capable of withstanding high turgor pressures and, simultaneously, that the PGN at their surface is concealed from receptors produced by the host innate immune system. For cell separation to occur, and for PGN to be kept concealed, “old” PGN is degraded by specific PGN hydrolases, also known as autolysins, that are found at the bacterial cell surface or that are secreted into the growth medium.
Bacterial PGN hydrolases are cell wall lytic enzymes that comprise a broad and diverse group of proteins. It is often difficult to assign a specific function to a ...
[摘要] 细菌细胞分裂期间细胞表面的代谢涉及细菌细胞壁的主要组分肽聚糖（PGN）的合成和降解。细菌必须确保它们的表面能够承受高的膨胀压力，同时，它们表面的PGN被宿主先天免疫系统产生的受体所掩盖。为了发生细胞分离，并且对于PGN保持隐蔽，“老”PGN由在细菌细胞表面发现或分泌到生长培养基中的特定PGN水解酶（也称为自溶素）降解。
细菌PGN水解酶是包含广泛和多样化蛋白质组的细胞壁溶解酶。主要是因为生物体可以具有大量具有冗余活性的水解酶，并且一种水解酶可以具有多种酶活性并参与各种细胞过程（Vollmer等，2008），通常难以将特定功能分配给PGN水解酶。 。枯草芽孢杆菌35种已知或假设的PGN水解酶，而大肠杆菌（E.coli）和金黄色葡萄球菌（金黄色葡萄球菌）分别具有约16和19个PGN水解酶（Vollmer，2012; Heidrich等，2001; Singh et al。 ，2012）。
PGN水解酶可分为三大类：糖苷酶，酰胺酶和肽酶。糖苷酶切割聚糖主链，分为N-乙酰氨基葡糖苷酶和N-乙酰神经酰胺酶。酰胺酶切割肽链和聚糖链的N-乙酰神经酰胺残基之间的连接。肽酶，如内肽酶和羧肽酶能够切割PGN干肽的不同氨基酸之间的肽键。
在这里，我们描述了提取与金黄色葡萄球菌细胞壁非共价连接的PGN水解酶的方法（Vollmer，2008）。含有变性PGN水解酶的提取物的分析是通过运行Zymogram凝胶（含有粗细菌细胞壁或底物细胞的SDS-PAGE凝胶）进行的，然后将其在非变性缓冲液中温育以允许PGN水解酶复性。然后可以通过产生在细胞壁消化发生时观察到的清除条带来鉴定这些复性酶。方案分为三个步骤：A）从金黄色葡萄球菌细胞中制备粗自动提取物; ...
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| In Gel Detection of Lipase Activity in Crude Plant Extracts (Olea europaea)
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Author:
Date:
2015-04-20
[Abstract] Here, we provide a detailed protocol describing a SDS-PAGE based procedure to assay in gel neutral lipase activity. Total protein extracts are separated by SDS-PAGE and gels are treated with lipase substrate-α-naphthyl palmitate. This long-chain fatty acid ester is hydrolysed by lipases present in the gel. The product resulting from this reaction can be then visualized in the gel as yellow-brownish activity bands. This relatively simple and effective method of lipase assay detection can be used for crude protein extracts from different plant tissues.
[摘要] 在这里，我们提供详细的协议描述基于SDS-PAGE的程序来测定凝胶中性脂肪酶活性。 通过SDS-PAGE分离总蛋白提取物，并用脂肪酶底物-α-棕榈酸萘酯处理凝胶。 这种长链脂肪酸酯被存在于凝胶中的脂肪酶水解。 由该反应产生的产物然后可以在凝胶中显现为黄棕色活性带。 这种相对简单和有效的脂肪酶测定法可用于来自不同植物组织的粗蛋白提取物。
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