| In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease
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Author:
Date:
2017-01-05
[Abstract] Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic clipping of histone tails is continuously attracting interest of researchers in the field of chromatin biology. We can recapitulate H3-clipping by performing in vitro H3 cleavage assay. Here, we are presenting the detailed protocol to perform in vitro H3 cleavage assay.
[摘要] 组蛋白受到广泛的可逆和不可逆的翻译后修饰（PTM）（Bannister和Kouzarides，2011; Azad和Tomar，2014）。已知组蛋白上的PTM调节染色质结构和功能。组蛋白不可逆地修饰其尾部结构域的蛋白水解剪切。组蛋白尾巴的蛋白水解剪切不断吸引研究人员在染色质生物学领域的兴趣。我们可以通过在体外实施H3切割测定来概括H3-剪切。在这里，我们提供了详细的方案来进行体外实验。
背景 组蛋白H3剪切是染色质修饰和调节最不了解的机制。预期H3剪切将永久性消除可能影响染色质相关事件的核小体的PTM。此外，切割的组蛋白的命运仍在研究之中，并且已经表明，切割的组蛋白可能在染色质的特定区域被再循环，或者它们被靶向降解。有各种各样的报告描述了不同生物中组蛋白H3的体内剪切，而组蛋白H3特异性剪切的体外测定是有限的。我们需要一种有效和稳健的体外实验来鉴定组蛋白特异性蛋白酶。为此，我们提出了一个可用于检查酵母和鸡肝组织蛋白H3蛋白酶的体外组蛋白H3剪切活性的方案。我们已经优化了测定的温度和pH条件。在我们优化的条件下，发现蛋白酶在所有核心组蛋白中特异性切割组蛋白H3。我们在最近的出版物（Chauhan等人，2016年; Chauhan和Tomar，2016年; Azad和Tomar，2016; ...
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| Extraction and Quantification of Polyphosphate in the Budding Yeast Saccharomyces cerevisiae
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Author:
Date:
2016-07-20
[Abstract] Inorganic polyphosphate (polyP) is a linear polymer present in both prokaryotic and eukaryotic organisms and made from three to hundreds of orthophosphate residues linked by phosphoanhydride bonds. The biological role of this molecule goes beyond serving as Pi store or energy source to replace ATP. For instance, in yeast polyP levels have been related to stress adaptation and this molecule has been shown to be the substrate for polyphosphorylation of proteins. Here we describe two different methods to purify polyP from the yeast Saccharomyces cerevisiae and the subsequent protocol to quantify polyP levels by spectrophotometrically measuring the Pi generated upon enzymatic hydrolysis of purified polyP. It must be noted that the purification protocol used greatly influences the ...
[摘要] 无机多磷酸盐（polyP）是存在于原核和真核生物中的线性聚合物，由通过磷酸酐键连接的三至数百个正磷酸盐残基制成。 这种分子的生物学作用超越了作为P 1储存或能量源以代替ATP。 例如，在酵母中，polyP水平与应激适应相关，并且该分子已经显示为蛋白质多磷酸化的底物。 在这里，我们描述了从酵母酿酒酵母中纯化polyP的两种不同方法和随后的通过分光光度法测量在酶水解纯化的polyP时产生的Pi来定量polyP水平的方案。 必须注意的是，所使用的纯化方案极大地影响所获得的polyP值。
图1. polyP的酶水解
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