| Kinetic Lactate Dehydrogenase Assay for Detection of Cell Damage in Primary Neuronal Cell Cultures
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Author:
Date:
2017-06-05
[Abstract] The aim of many in vitro models of acute or chronic degenerative disorders in the neurobiology field is the assessment of survival or damage of neuronal cells. Damage of cells is associated with loss of outer cell membrane integrity and leakage of cytoplasmic cellular proteins. Therefore, activity assays of cytoplasmic enzymes in supernatants of cell cultures serve as a practicable tool for quantification of cellular injury (Koh and Choi, 1987; Bruer et al., 1997). Lactate dehydrogenase (LDH) is such a ubiquitously expressed cytosolic enzyme, which is very stable due to a very long protein half-life (Hsieh and Blumenthal, 1956; Koh and Cotman, 1992; Koh et al., 1995).
[摘要] 神经生物学领域中许多急性或慢性退行性疾病的体外模型的目的是评估神经元细胞的存活或损伤。细胞损伤与外细胞膜完整性的丧失和细胞质细胞蛋白的泄漏有关。因此，细胞培养上清液中细胞质酶的活性测定作为细胞损伤定量的实用工具（Koh和Choi，1987; Bruer等，1997）。乳酸脱氢酶（LDH）是一种无处不在表达的细胞溶质酶，由于蛋白质的半衰期很长，其稳定性很高（Hsieh和Blumenthal，1956; Koh和Cotman，1992; Koh等人）。 ，1995）。
背景 LDH在可逆的生物化学反应中催化丙酮酸和还原的烟酰胺偶氮二核苷酸（NADH）的乳酸盐和烟酰胺氨基茚三核苷酸（NAD +）的形成。 NADH在340nm的波长上具有吸收。这种动力学LDH活性测定的基础是由NADH降低引起的特定波长处的光密度降低。使用具有已知LDH活性的标准酶溶液计算上清液中LDH的量。不同的细胞密度或代谢活化率可能是混杂的;因此推荐LDH活性的正常化。这是通过评估外部细胞膜裂解后不抑制LDH活性的LDH活性（用0.5％Triton-X“完全杀死”）来实现的。最后，通过完全杀死的LDH活性的绝对LDH活性百分比表示细胞培养物中损伤或死细胞的发生率。
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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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| A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD+/NADH in Streptococcus mutans
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Author:
Date:
2016-08-20
[Abstract] Nicotinamide adenine dinucleotide is a coenzyme present in all kingdoms of life and exists in two forms: oxidized (NAD+) and reduced (NADH). NAD(H) is involved in a multitude of essential metabolic redox reactions, providing oxidizing or reducing equivalents. The ratio of free intracellular NAD+/NADH is fundamentally important in the maintenance of cellular redox homeostasis (Ying, 2008). Various chromogenic cycling assays have been used to determine the ratio of NAD+/NADH in both bacterial and mammalian cells for more than forty years (Bernofsky and Swan, 1973; Nisselbaum and Green, 1969).
Here, we describe in detail an assay to determine the ratio of free intracellular NAD+ to NADH in Streptococcus mutans. This cycling ...
[摘要] 烟酰胺腺嘌呤二核苷酸是存在于生命的所有王国中的辅酶，并且以两种形式存在：氧化（NAD +）和还原（NADH）。 NAD（H）参与多种必需的代谢氧化还原反应，提供氧化或还原等价物。游离细胞内NAD + /NADH的比率在维持细胞氧化还原稳态中是根本重要的（Ying，2008）。已经使用各种显色循环测定来测定细菌和哺乳动物细胞中NAD +/NAD +/NADH的比率超过四十年（Bernofsky和Swan，1973; Nisselbaum和Green，1969）。 /> 在这里，我们详细描述了测定变异链球菌中游离细胞内NAD + 与NADH的比率的测定法。该循环测定是Bernofsky和Swan（Bernofsky和Swan，1973）首先描述的方案的修改版本，使用Frezza等人描述的提取缓冲液。 （2011），随后是由Gibbon和Larher描述的减少的MTT降水（Gibon和Larher，1997）。如图1所示，使用乙醇脱氢酶来驱动一系列氧化还原反应，利用来自样品提取物的外源添加的乙醇和NAD +作为初始底物，吩嗪乙硫酸盐（PES）作为电子载体，以及噻唑基蓝色四唑溴化物（MTT）作为末端电子受体。使用6M ...
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