| Expression, Purification and Enzymatic Assay of Plant Histone Deacetylases
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Author:
Date:
2016-04-05
[Abstract] Histone deacetylases (HDACs) catalyzing the removal of acetyl groups from lysine residues of histone and non-histone proteins play vital roles in regulation of gene transcription. In plants, HDACs can be grouped into three families, including RPD3-type, SIR2-type and plant specific HD2-type HDACs. Here we describe a method to determine plant HDAC enzymatic activity. This protocol includes expression, purification and enzymatic activity assay of recombinant plant HDACs expressed in Escherichia coli (E. coli) and Arabidopsis thaliana (A. thaliana).
[摘要] 组蛋白脱乙酰酶（HDAC）催化从组蛋白和非组蛋白蛋白的赖氨酸残基去除乙酰基在调节基因转录中起重要作用。 在植物中，HDAC可以分为三个家族，包括RPD3型，SIR2型和植物特异性HD2型HDAC。 在这里我们描述了一种确定植物HDAC酶活性的方法。 该方案包括在大肠杆菌（大肠杆菌）和拟南芥（）中表达的重组植物HDAC的表达，纯化和酶活性测定 >拟南芥）。
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| Assays of Polyphenol Oxidase Activity in Walnut Leaf Tissue
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Author:
Date:
2014-08-20
[Abstract] Polyphenol oxidase (PPO) is an enzyme that catalyzes the hydroxylation of monophenols into ortho-diphenols (cresolase activity) and the oxidation of o-diphenols into quinones (catecholase activity) (Figure 1). These quinones spontaneously polymerize to form dark-colored phytomelanins, most often seen in the browning of damaged plant tissue. PPO activity can be easily assayed in crude protein extracts from English walnut (Juglans regia) leaves and from many other plant tissue extracts. PPO activity is most commonly measured by spectrophotometric assay, in which the rate of phytomelanin production is quantified, or by oxygen electrode assay, in which the consumption of oxygen by the enzyme is quantified (Figure 1). Though simpler, the utility of the spectrophotometric ...
[摘要] 多酚氧化酶（PPO）是催化单酚羟基化成邻二酚（甲酚酶活性）和将β-二酚氧化成醌（儿茶酚酶活性）（图1）的酶。这些醌自发聚合形成深色的植物色素，最常见于受损的植物组织的褐变。 PPO活性可以容易地在来自英国胡桃（Juglans regia）叶和来自许多其它植物组织提取物的粗蛋白提取物中测定。 PPO活性最通常通过分光光度测定法测量，其中植物肉豆蔻毒素产生的速率被量化，或通过氧电极测定，其中酶对氧的消耗量化（图1）。尽管更简单，分光光度测定的效用受到从不同酚类底物产生的植物角蛋白的最大吸收的变化的限制。氧电极测定通常被认为是用于测量PPO活性的"金标准"，但是与单酚底物一起实施是更费时和困难的，因为与儿茶酚酶活性相比，甲酚水解酶活性通常相当低。该方案将描述从核桃叶的粗蛋白质提取，分光光度测定法和用于测定PPO活性的氧电极测定法。
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