| Extraction and Assays of ADP-glucose Pyrophosphorylase, Soluble Starch Synthase and Granule Bound Starch Synthase from Wheat (Triticum aestivum L.) Grains
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Author:
Date:
2016-09-20
[Abstract] Starch biosynthesis in plants involves a network of enzymes of which adenosine-5’-diphosphoglucose (ADP-glucose) pyrophosphorylase (AGPase, E.C. 2.7.7.27), and soluble and granule bound starch synthases (SSS and GBSS, E.C. 2.4.1.21) play central roles. Here, we outline the protocol for extraction and assay of these enzymes in developing grains of wheat (Triticum aestivum L.). The principle of the assays outlined is based on a coupling enzymatic reactions where the product of the initial reaction is used as a substrate for subsequent reactions in order to generate NADPH, which can be measured easily by spectrophotometer. This protocol does not need expensive labelled chemicals and can be carried out using equipment commonly found in a biochemical laboratory. We applied this ...
[摘要] 植物中的淀粉生物合成涉及酶的网络，其中腺苷-5'-二磷酸葡萄糖（ADP-葡萄糖）焦磷酸化酶（AGPase，EC 2.7.7.27）和可溶性和颗粒结合的淀粉合酶（SSS和GBSS，EC 2.4.1.21）中心角色。在这里，我们概述了用于提取和测定这些酶在开发小麦谷粒（ Triticum aestivum L）中的方案。概述的测定法的原理基于偶联酶反应，其中初始反应的产物用作随后反应的底物，以便产生NADPH，其可以通过分光光度计容易地测量。该方案不需要昂贵的标记的化学品，并且可以使用通常在生化实验室中发现的设备进行。我们应用这个协议来研究开花后不同时间点小麦籽粒中AGPase，SSS和GBSS活性的动力学。
[背景] 淀粉是一种碳水化合物聚合物由α-1,4-连接的葡萄糖分子组成的线性葡聚糖聚合物和支链淀粉（由α-1,6-糖苷键支化的α-1,4-连接的葡萄糖分子组成的另一种葡聚糖聚合物）。腺苷-5'-二磷酸葡萄糖（ADP-葡萄糖）焦磷酸化酶（AGPase，E.C.2.7.7.27）催化淀粉合成的第一承诺步骤，将葡萄糖-1-磷酸和ATP转化为ADP-葡萄糖和无机焦磷酸（PPi）。 ADP-葡萄糖随后被可溶性淀粉合成酶（SSS）和颗粒结合的淀粉合酶（GBSS）（EC 2.4.1.21）和淀粉分支酶使用以延长和分支淀粉颗粒的葡聚糖链。 ...
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| Measurement of Proton-driven Antiport in Escherichia coli
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Author:
Date:
2014-11-05
[Abstract] Secondary active transport of substrates across the inner membrane is vital to the bacterial cell. Of the secondary active transporter families, the ubiquitous major facilitator superfamily (MFS) is the largest and most functionally diverse (Reddy et al., 2012). Recently, it was reported that the MFS multidrug efflux protein MdtM from Escherichia coli (E. coli) functions physiologically in protection of bacterial cells against bile salts (Paul et al., 2014). The MdtM transporter imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an antiport reaction. This protocol describes, using fluorometry, how to detect the bile salt/H+ ...
[摘要] 底物穿过内膜的二次主动转运对细菌细胞是至关重要的。在次要活性转运蛋白家族中，遍在的主要促进子超家族（MFS）是最大和最功能多样的（Reddy等人，2012）。最近，据报道，来自大肠杆菌（大肠杆菌）的MFS多药物外排蛋白MdtM在保护细菌细胞对抗胆汁盐中具有生理功能（Paul等人al。，2014）。 MdtM转运蛋白通过将外部质子（H + +）的交换耦合到通过反向端反应从细胞内部排出的胆汁盐，赋予细菌细胞以抗胆汁盐性。该方案使用荧光测定法描述了如何在E的逆向转运体缺陷菌株的倒置膜囊泡中检测MdtM的胆汁盐/H sup/+抗末端活性。通过测量跨膜ΔpH测定大肠杆菌 TO114细胞。该方法利用pH敏感染料吖啶橙的荧光信号（淬灭和去淬灭）的强度响应于囊泡腔中的[H sup +]的变化而发生的变化。由于内源性转运蛋白表达的低水平，其通常使得单个转运蛋白例如MdtM对质子驱动的反运输蛋白的贡献难以检测，所述方法通常需要从多拷贝质粒过表达所关注的转运蛋白。尽管本文所述的方案的第一部分对于来自pBAD/emyc-His-A表达载体的MdtM的过表达非常特异，但描述随后通过MdtM测量胆汁盐流出的方案可以容易地适用于通过任何其他反交换器测量其它底物的反向运输，其交换质子用于对衬底。
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| Measurement of the Electrogenicity of Bile Salt/H+ Antiport in Escherichia coli
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Author:
Date:
2014-11-05
[Abstract] The transmembrane proton gradient (ΔpH) is the primary source of energy exploited by secondary active substrate/H+ antiporters to drive the electroneutral transport of substrates across the Escherichia coli (E. coli) inner membrane. Such electroneutral transport results in no net movement of charges across the membrane. The charge on the transported substrate and the stoichiometry of the exchange reaction, however, can result in an electrogenic reaction which is driven by both the ΔpH and the electrical (∆Ψ) components of the proton electrochemical gradient, resulting in a net movement of electrical charges across the membrane. We have shown that the major facilitator superfamily transporter MdtM - a multidrug efflux protein from E. coli that ...
[摘要] 跨膜质子梯度（ΔpH）是由次级活性底物/H sup +反转录子开发的能量的主要来源，以驱动底物穿过大肠杆菌的电中性转运（ >大肠杆菌）内膜。这种电中性转运导致电荷没有跨膜的净移动。然而，运输的底物上的电荷和交换反应的化学计量可以导致由ΔpH和质子电化学梯度的电（ΔΨ）分量驱动的电致反应，导致电移动的净移动电荷穿过膜。我们已经显示主要促进子超家族转运蛋白MdtM-来自E的多药物外排蛋白。在保护细菌细胞对抗胆汁盐中起到生理作用的大肠杆菌通过偶联外部质子（H + +）与胆汁盐的外流的交换而赋予细菌细胞对胆汁盐的抗性通过电致反应反应的细胞内部（Paul et al。，2014）。该协议使用荧光测定法描述了如何检测Ed的逆转子缺陷菌株的倒置膜囊泡中MdtM的电致逆转运蛋白活性。通过测量跨膜ΔΨ测定大肠杆菌 TO114细胞。该方法利用响应于由于MdtM催化的胆酸钠/H +交换反应引起的膜电位变化而发生在探针Oxonol V的荧光信号强度（淬灭和去淬灭）中发生的变化。该方案可以适于检测任何辅助活性反转运蛋白的活性，其将H sup +跨越生物膜的电致易位与其对应底物的电转运偶联，并且可以用于解偶联否则伪装的转运活性和生理作用。
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