| Assay of Arabinofuranosidase Activity in Maize Roots
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Author:
Date:
2016-03-20
[Abstract] Root is a perfect model for studying the mechanisms of plant cell growth. Along the root length, several zones where cells are at different stages of development can be visualized (Figure 1). The dissection of the root on these zones allows the investigation of biochemical and genetic aspects of different growth steps. Maize primary root is much more massive than the root of other Monocots and thus more convenient for such type of research. Plant cell wall, mainly consisting of polysaccharides, plays an important role in plant life. Therefore, measurement of plant carbohydrate content and glycoside-modifying enzyme activity in plant cells has become an important aspect in plant physiology. One of the well-documented changes of hemicelluloses molecules during elongation growth of monocots ...
[摘要] 根是研究植物细胞生长机制的完美模型。沿着根长度，可以可视化细胞处于不同发育阶段的几个区（图1）。在这些区域上的根的解剖允许调查不同生长步骤的生化和遗传方面。玉米主根比其他单根的根更大，因此这种类型的研究更方便。植物细胞壁，主要由多糖组成，在植物生活中发挥重要作用。因此，植物碳水化合物含量和糖苷修饰酶活性在植物细胞中的测量已经成为植物生理学中的重要方面。在单子叶植物细胞的延长生长期间半纤维素分子的充分证明的变化之一是葡糖醛酸阿拉伯木聚糖的阿拉伯糖取代的减少。这可能是由于该多糖的合成的变化或阿拉伯呋喃糖苷酶的作用引起的。在这里，我们描述的分光光度测量阿拉伯呋喃糖苷酶活性在玉米根中的水解发色底物（4-硝基苯基α-L-阿拉伯呋喃糖苷）的速率的协议。
图1. 四日龄黑暗生长的玉米幼苗面板）。根据Kozlova等人（2012）（右图），初级玉米根的不同区和细胞发育的相应阶段。
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| Assays for Determination of Acetylesterase Activity and Specificity Using pNP-acetyl and Acetylated Polysaccharides as Substrates
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Author:
Date:
2014-02-05
[Abstract] The acetylesterases are hydrolytic enzymes which in plants cleave acetyl groups from acetylated cell wall components, primarily polysaccharides. To estimate acetylesterase activity in plant apoplast, two assays can be used. First assay is a direct measurement of the acetylesterase activity in protein extract using synthetic substrate, pNP-acetyl. In this assay, amount of pNP released after hydrolysis of pNP-acetyl is determined by measuring the intensity of developed yellow color using spectrophotometer. The absorbance of reaction mixture is directly proportional to the activity of acetylesterases in the reaction mixture. Second assay is a determination of acetylesterase activity and its specificity towards natural polysaccharides and based on interaction between ferric perchlorate and ...
[摘要] 乙酰酯酶是水解酶，其在植物中从乙酰化的细胞壁组分，主要是多糖中切割乙酰基。为了估计植物质外体中的乙酰酯酶活性，可以使用两种测定。第一个测定是使用合成底物，pNP-乙酰基直接测量蛋白质提取物中的乙酰酯酶活性。在该测定中，通过使用分光光度计测量显色黄色的强度来确定pNP-乙酰基水解后释放的pNP的量。反应混合物的吸光度与反应混合物中乙酰酯酶的活性成正比。第二个测定是乙酰酯酶活性及其对天然多糖的特异性的测定，并且基于高氯酸铁和乙酰基残基之间的相互作用，导致可以使用分光光度计定量的乙酰氧肟酸络合物。在该测定中，使用高粱酸铁试剂来估计与质外体提取物孵育的市售乙酰化多糖（来自Birchwood的木聚糖用于乙酰木聚糖酯酶的果胶;来自柑橘属水果的用于鼠李半乳糖醛酸聚糖乙酰酯酶的果胶;或任何其他可用的目的多糖）和量的从该多糖中释放的乙酰基残基，试剂（方案由McComb和McCready，1957修改）。产生的有色复合物的吸光度与从乙酰化多糖释放的乙酰基的量成正比。
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