| 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 ...
[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 ...
[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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| 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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