| Measurement of ATP Hydrolytic Activity of Plasma Membrane H+-ATPase from Arabidopsis thaliana Leaves
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Author:
Date:
2016-12-05
[Abstract] Plant plasma membrane H+-ATPase, which is a P-type ATPase, couples ATP hydrolysis to H+ extrusion and thereby generates an electrochemical gradient across the plasma membrane. The proton gradient is necessary for secondary transport, cell elongation, and membrane potential maintenance. Here we describe a protocol for measurement of the ATP hydrolytic activity of the plasma membrane H+-ATPase from Arabidopsis thaliana leaves.
[摘要] 作为P型ATP酶的植物质膜H sup + -ATPase将ATP水解耦合到H + +/- 挤出，从而在质膜上产生电化学梯度。质子梯度对于二次转运，细胞伸长和膜电位维持是必需的。这里我们描述用于测量来自拟南芥叶片的质膜H + -ATPase的ATP水解活性的方案。
关键词： strong> 拟南芥，ATP水解活性，原钒酸盐，P-型ATP酶，血浆膜H + -ATPase
质膜H + -ATPase活性的测定对于阐明其功能和调节机制是重要的。然而，有时难以确定质膜H sup + -ATP酶的ATP水解活性，因为植物细胞含有许多ATP水解酶。该协议是基于Uemura和Yoshida（1986）和Kinoshita等人的出版物开发的。 （1995）。我们使用KNO 3作为V型ATP酶的抑制剂，钼酸铵作为酸性磷酸酶的抑制剂，寡霉素作为F型ATP酶的抑制剂，NaF作为磷酸酶的抑制剂（Shimazaki和Kondo ，1987; Kinoshita等人，1995）。原钒酸盐抑制P型ATP酶，并且因此可以通过评估来自ATP水解的钒酸盐敏感性P 1释放来用于测量质膜H sup + -ATP酶的活性。释放的P 1与钼酸盐反应形成蓝色络合物，然后可以通过测量在750nm的吸收来量化。
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| Differentiation of Naturally Produced Extracellular Membrane Vesicles from Lipid Aggregation by Glucuronoxylomannan Immunogold Transmission Electron Microscopy in Bacillus subtilis
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Author:
Date:
2015-03-05
[Abstract] Recently, membrane vesicle (MV) production was described in Gram-positive bacteria, which harbor a variety of components such as toxins, antibiotic resistance proteins, proteases, DNA, and immune modulators. Free lipids have the ability to form micelles, thus it is important to rule out spontaneous association of lipids into vesicle-like structures and rather, that MVs are produced naturally by a metabolically active cell. Here, we describe a protocol utilizing the polysaccharide, glucuronoxylomannan (GXM) from Cryptococcus neoformans (C. neoformans) as a marker to ...
[摘要] 最近，在革兰氏阳性细菌中描述了膜囊泡（MV）产生，其含有多种组分，例如毒素，抗生素抗性蛋白，蛋白酶，DNA和免疫调节剂。游离脂质具有形成胶束的能力，因此排除脂质自发缔合成囊泡样结构是重要的，并且更确切地说，MV由代谢活性细胞天然产生。在这里，我们描述了利用来自新隐孢子虫的多糖葡萄糖醛酸甘露聚糖（GXM）（ C。新型隐球菌）作为标记物来区分天然产生的MV与自发形成的囊泡革兰氏阳性模式生物，枯草芽孢杆菌（<枯草芽孢杆菌）。从在GXM存在下生长的细菌培养物中纯化MV;由细胞天然产生的MV在管腔中不含有GXM，而在培养基中形成的囊泡结构可包封GXM，并且这可通过免疫金属透射电子显微镜可视化。
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| Vesicle Isolation from Bacillus subtilis Biofilm
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Author:
Date:
2015-03-05
[Abstract] Bacterial biofilms are associated clinically with many bacterial infections including those caused by bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. In recent years, extracellular vesicles produced by bacteria have been isolated from biofilm communities. Vesicles have been described in depth and can encapsulate various virulence factors including toxins and immunomodulatory compounds. Vesicles may be important for virulence and survival by serving as a vehicle for the secretion and concentrated delivery of these molecules. Studying extracellular vesicles ...
[摘要] 细菌生物膜临床上与许多细菌感染相关，包括由细菌如绿脓杆菌和金黄色葡萄球菌引起的细菌感染。 近年来，由细菌产生的胞外囊泡已经从生物膜群落中分离。 囊泡已经被深入描述并且可以包封各种毒力因子，包括毒素和免疫调节化合物。 通过作为这些分子的分泌和浓缩递送的载体，囊泡可能对于毒力和存活是重要的。 研究胞外囊泡是理解生物膜形成，结构和破裂的重要步骤，其最终目的是预防或治疗由存在于生物膜中的细菌病原体引起的医院感染。 在这里我们描述了从枯草芽孢杆菌生产的生物膜中分离囊泡的方案。
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