| Determination of Reduced and Total Glutathione Content in Extremophilic Microalga Galdieria phlegrea
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Author:
Date:
2017-07-05
[Abstract] Glutathione is an important molecule involved in the primary and secondary metabolism of all organisms. The Glutathione redox status is an indicator of the cellular redox state. Therefore, it is important to have precise methods on hand to determine the glutathione redox status in the cell. In this protocol, we describe an improved spectrophotometric method to estimate the content of reduced (GSH) and oxidized (GSSG) forms of glutathione in the extremophilic microalga Galdieria phlegrea.
[摘要] 谷胱甘肽是涉及所有生物体的初级和次级代谢的重要分子。 谷胱甘肽氧化还原状态是细胞氧化还原状态的指标。 因此，重要的是要有精确的方法来确定细胞中的谷胱甘肽氧化还原状态。 在本协议中，我们描述了一种改进的分光光度法，用于估计极地微藻Galdieria phlegrea中还原（GSH）和氧化（GSSG）形式的谷胱甘肽的含量。
【背景】谷胱甘肽（γ-L-谷氨酰基-L-半胱氨酰 - 甘氨酸）是存在于所有已知生物体中的必需三肽，其范围从细菌到人类（Frendo等人，2013）。它涉及到几种不同的细胞保护作用（图1）。谷胱甘肽被认为是具有氧化还原信号功能的优异的抗氧化分子。它还参与对非生物和生物胁迫的反应，并涉及细胞的初级代谢（C，N，S代谢）（Noctor et al。，2012;Hernándezet al。 ，2015; Salbitani等人，2015）。
＆NBSP;在植物细胞中，还原型和氧化形式的谷胱甘肽（GSH / GSSG）之间的比例在信号传导和许多防御机制的激活中起重要作用（Foyer和Noctor，2012; Salbitani等人， ，2015）。通常严格控制的GSH / GSSG氧化还原状态可能在应力条件下瞬时转向稍微氧化的值（Tausz等人，2004）。
＆NBSP;在过去，一些研究人员已经开发和修改了分光光度法来确定几种生物体中的GSH和GSSG（Anderson，1985; ...
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| Determination of Recombinant Mannitol-1-phosphate Dehydrogenase Activity from Ectocarpus sp.
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Author:
Date:
2016-11-05
[Abstract] Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of the recombinant catalytic domain of one ...
[摘要] 褐藻属于与绿色植物和动物遥远相关的系统发生谱系，并且主要发现于但不限于潮间带，一种苛刻且频繁变化的环境。由于它们独特的进化史和它们的栖息地，褐藻具有其代谢中的一些特性。其中之一是甘露醇循环，其在其生理学中发挥中心作用，因为甘露糖醇充当碳储存，渗透保护剂和抗氧化剂。该多元醇通过甘露醇-1-磷酸酯脱氢酶（M1PDH，EC 1.1.1.17）和甘露醇-1-磷酸酶（M1Pase，EC 3.1.3.22）的作用直接从光生酸酯果糖-6-磷酸衍生。该协议描述了在Ectocarpus中鉴定的三种M1PDH之一的重组催化结构域的生物化学表征。该重组催化结构域（下文称为M1PDHcat）使用NAD（H）作为辅因子催化果糖-6-磷酸（F6P）向甘露醇-1-磷酸（M1P）的可逆转化。 M1PDHcat活性在两个方向上测定，即，F6P还原和M1P氧化（图1）。
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图1.甘露醇-1-磷酸脱氢酶的可逆反应
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| Determination of Mannitol-2-dehydrogenase Activity
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Author:
Date:
2015-11-05
[Abstract] Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. Several pathways have been described for the metabolism of mannitol by bacteria, including the phosphoenolpyruvate-dependent phosphotransferase system (PST) and a M2DH-based catabolic pathway. The latter involves two enzymes, a mannitol-2-dehydrogenase (EC 1.1.1.67) and a fructokinase (EC 2.7.1.4), and has been identified in different bacteria, e.g., the marine Bacteroidetes Zobellia galactanivorans (Zg) which had recently gained interest to study the degradation of macroalgal polysaccharides. This protocol describes the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) of Zobellia galactanivorans. The Zg ...
[摘要] 甘露醇是在多种活生物体中发生的多元醇，其中它实现不同的生理作用。已经描述了用于由细菌代谢甘露醇的几种途径，包括磷酸烯醇丙酮酸依赖性磷酸转移酶系统（PST）和基于M2DH的分解代谢途径。后者涉及两种酶，甘露醇-2-脱氢酶（EC 1.1.1.67）和果糖激酶（EC 2.7.1.4），并且已经在不同的细菌中鉴定，例如， 海洋拟杆菌（Zobellia galactanivorans）（ Zg ），最近对研究大分子多糖降解有兴趣。该方案描述了半乳聚糖微生物的重组甘露醇-2-脱氢酶（M2DH）的生物化学表征。使用NAD +辅助作为辅因子，M2DH酶催化甘露醇向果糖的可逆转化。 在两个方向上测定M2DH活性，即果糖还原和甘露醇氧化。
可逆反应：
D-甘露醇+ NAD + ↔D-果糖+ NADH + H +
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