| Determination of the H+-ATP Synthase and Hydrolytic Activities
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Author:
Date:
2016-08-20
[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。
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| Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels
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Author:
Date:
2014-08-05
[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。
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