| 59Fe Uptake Assays in Paracoccidioides Species
|
|
Author:
Date:
2016-09-20
[Abstract] Iron is an essential micronutrient required for virtually all organisms. This fact is related to the ability of the transition metal to exist in two oxidation states, the reduced ferrous (Fe2+) and the oxidized ferric (Fe3+). Given the relative availability of aqueous iron (the element which constitutes ~5% of the earth’s crust) one is not surprised that iron is the most common prosthetic element in biology. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA) (Kosman, 2013). In this way, the uptake of iron in the absence or presence of the reducing agent ascorbic acid can be investigated by 59Fe uptake assays, as previously described (Eide et al., 1992). In the ...
[摘要] 铁是几乎所有生物体所必需的微量营养素。这一事实涉及过渡金属以两种氧化态存在的能力，即还原的亚铁（Fe 2+）和氧化的铁（Fe 3++）。考虑到铁水（构成地壳的〜5％的元素）的相对可用性，人们不惊讶的是，铁是生物学中最常见的假体元素。通常，真菌可以通过受体介导的铁载体或血红素的内化，和/或还原铁同化（RIA）吸收铁（Kosman，2013）。以这种方式，如上所述，可以通过59 Fe吸收测定来研究在还原剂抗坏血酸存在或不存在下铁的吸收（Eide等人）。 ，1992）。在抗坏血酸存在下，研究还原非依赖性59 Fe吸收途径。另一方面，在不存在抗坏血酸的情况下，刺激还原依赖性59 Fe吸收途径。使用这种策略用于人类致病真菌Paracoccidioides物种，结果表明，在没有抗坏血酸的情况下，通过 01的铁摄取是低的，不同于在 > Pb 18。这些结果表明，只有在Fe 18中，铁摄取路径与铁还原酶偶联（Bailão等人，2015）。在该方案中，我们描述了如何在Paracoccidioides物种中进行 59 Fe摄取测定。
|
|
|
| Determination of the H+-ATP Synthase and Hydrolytic Activities
|
|
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。
|
|
|