{{'Search' | translate}}

Adenosine 5′-diphosphate sodium salt


Company: Sigma-Aldrich
Catalog#: A2754
Other protocol()

Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells
[Abstract]  Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and Wilmore, 1990). Glutamine is involved in many biological processes including serving as the nitrogen donor for biosynthesis, as an exchanger for the import of essential amino acids, as a means to detoxifying intracellular ammonia and glutamate, and as a bioenergetics nutrient to fuel the tricarboxylic acid (TCA) cycle (Bott et al., 2015). The method for the assay of GS enzymatic activity relies on its γ-glutamyl transferase reaction by ... [摘要]  谷氨酰胺合成酶(GS),其催化谷氨酸和氨转化成谷氨酰胺,广泛分布在动物组织和细胞培养系中。该酶的重要性通过谷氨酰胺,GS-催化的从头合成反应的产物,是血液中最丰富的游离氨基酸的事实提示(Smith和Wilmore,1990)。谷氨酰胺参与许多生物过程,包括作为生物合成的氮供体,作为输入必需氨基酸的交换剂,作为解毒细胞内氨和谷氨酸的手段,以及作为生物能量营养物来给三羧酸(TCA)周期(Bott等人,2015)。用于测定GS酶活性的方法依赖于其γ-谷氨酰转移酶反应,通过测量由谷氨酰胺和羟胺合成的γ-谷氨酰羟肟酸酯,以及反应产物与反应物的色谱分离(Deuel等人 。,1978)。 GS谷氨酰转移酶反应的概述可以在图1中找到。通过分光光度测定法在560nm的特定波长下使用酶标仪测量GS活性。该方法简单,并且具有与应用放射性标记的底物的那些方法相当的灵敏度。该修改的方法已经应用于在包括人乳腺上皮MCF10A细胞和鼠前B FL5.12细胞的培养细胞系中测定/测定GS活性,并且可以用于测量其他细胞系中的GS活性。 >

图1 。GS glutamyl ...

Determination of the H+-ATP Synthase and Hydrolytic Activities
[Abstract]  The H+-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions, it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and hydrolytic activities of the H+-ATP synthase in isolated mitochondria and in permeabilized mammalian cells. Since the protocol requires the isolation of polarized and well-coupled mitochondria, first we describe the protocol for mitochondrial isolation from mouse tissues. Second, we describe the protocol for measuring the ATP synthetic activity as end-point and kinetic modes in isolated mitochondria and in permeabilized cells. Finally, we ... [摘要]  内线粒体膜的H sup + -ATP合酶利用呼吸链产生的质子梯度来合成ATP。 在去极化条件下,它可以通过水解ATP产生质子梯度而反向作用。 本文提供的方案允许容易地测定分离的线粒体和透化的哺乳动物细胞中H sup + -ATP合酶的合成和水解活性。 由于该协议需要极化和良好耦合的线粒体的分离,首先我们描述线粒体从小鼠组织分离的协议。 第二,我们描述了用于测量ATP合成活性作为终点和在分离的线粒体和透化细胞中的动力学模式的方案。 最后,我们描述了用于测定酶在分离的线粒体中的ATP水解活性的方案。

Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels
[Abstract]  It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays. [摘要]  最近报道了番茄果实色素可以使用含有非典型γ亚基的ATP合酶复合物来合成ATP,所述亚基也存在于多种植物物种中。 然而,与这个过程基础的生化过程相关的许多方面仍然很大程度上未知。 在这里我们描述了隔离番茄果实色质体和ATP水平(终点测量)和ATP合成率(动力学测量)在这些细胞器中使用基于生物发光荧光素/荧光素酶的测定的确定详细协议。