| Metabolic Heavy Isotope Labeling to Study Glycerophospholipid Homeostasis of Cultured Cells
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Author:
Date:
2017-05-05
[Abstract] Glycerophospholipids consist of a glycerophosphate backbone to which are esterified two acyl chains and a polar head group. The head group (e.g., choline, ethanolamine, serine or inositol) defines the glycerophospholipid class, while the acyl chains together with the head group define the glycerophospholipid molecular species. Stable heavy isotope (e.g., deuterium)-labeled head group precursors added to the culture medium incorporate efficiently into glycerophospholipids of mammalian cells, which allows one to determine the rates of synthesis, acyl chain remodeling or turnover of the individual glycerophospholipids using mass spectrometry. This protocol describes how to study the metabolism of the major mammalian glycerophospholipids i.e., phosphatidylcholines, ...
[摘要] 甘油磷脂由甘油磷酸酯骨架组成，酯基化两个酰基链和极性头基。头组（例如胆碱，乙醇胺，丝氨酸或肌醇）限定了甘油磷脂类，而酰基链与头基一起限定了甘油磷脂分子种类。添加到培养基中的稳定的重质同位素（例如，氘）标记的头基前体有效地并入哺乳动物细胞的甘油磷脂中，这允许人们确定合成速率，酰基链重塑或周转使用质谱法测定个体甘油磷脂。该方案描述了如何用这种方法研究主要哺乳动物甘油磷脂的代谢，即磷脂酰胆碱，磷脂酰乙醇胺，磷脂酰丝氨酸和磷脂酰肌醇。
背景 放射性标记的前体已广泛用于研究培养细胞中的甘油磷脂（GPL）代谢。然而，这种方法有严重的缺点。首先，研究GPL的所有分子种类的代谢是不可行的，因为不可能的事实，即没有恢复到高度复杂和耗时的方案来将各个分子种类彼此分离（Patton＆ em等人，1982），这显然是研究其新陈代谢所必需的。第二，所需的放射性同位素相当昂贵。第三，为了最佳标记，未标记的前体应尽可能耗尽介质。第四，可以同时向细胞中加入两种不同的前体，即使对同位素光谱之间的重叠进行准确校正是必要的对所收集的部分进行液体闪烁计数。由于这些障碍，许多研究最近引入了研究GPL代谢的替代方法（例如，Heikinheimo和Somerharju，2002; de Kroon，2007; Kainu等人2008年; Postle和Hunt，2009; ...
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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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| Separation of Microspores from Anthers of Lilium longiflorum (Lily) and Subsequent RNA Extraction
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Author:
Date:
2015-02-20
[Abstract] This protocol has been designed in order to facilitate the isolation and extraction of total RNA from microspores collected from lily anther sacs. This protocol allows the extraction of high amounts of high quality RNA, as observed in agarose gels.
[摘要] 该协议已被设计以便于从从百合花囊收集的小孢子中分离和提取总RNA。 这个协议允许大量的高质量RNA，如在琼脂糖凝胶中观察到的提取。
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