Dove Stephen K, Michell Robert H
Phosphoinositide Lab, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
Methods Mol Biol. 2009;462:59-74. doi: 10.1007/978-1-60327-115-8_4.
Inositol phospholipids regulate many cellular processes, including cell survival, membrane trafficking, and actin polymerization. Quantification of inositol lipids is one of the essential techniques needed for studies that aim to decipher inositol lipid-dependent cellular functions. The study of phosphoinositides in most organisms is hampered by a lack of facile genetic tools. However, the essential elements of most inositol lipid signaling pathways appear to be conserved across eukaryote phylogeny. They can therefore readily be elucidated (both genetically and biochemically) in the budding yeast Saccharomyces cerevisiae. Because of the low abundance of polyphosphoinositides in cells, many analytical methods start by radioactively labeling intact cells and then extracting the lipids with chloroform/methanol/ water mixtures based on those first devised half a century ago. Yeast present special extraction problems because the cell wall must be broken in order to facilitate solvent access and maximize lipid yield. Once lipids have been extracted, fatty acids are removed and the resulting water-soluble glycerophosphoinositol phosphates are analysed by anion-exchange HPLC. This chapter describes how to extract and quantify the inositol lipids of S. cerevisiae cells that have been radiolabeled to isotopic equilibrium with [3H]myo-inositol.
肌醇磷脂调节许多细胞过程，包括细胞存活、膜运输和肌动蛋白聚合。肌醇脂质的定量分析是旨在破译肌醇脂质依赖性细胞功能的研究所必需的关键技术之一。在大多数生物体中，磷酸肌醇的研究因缺乏便捷的遗传工具而受到阻碍。然而，大多数肌醇脂质信号通路的基本元件在真核生物系统发育中似乎是保守的。因此，它们可以在出芽酵母酿酒酵母中很容易地（通过遗传学和生物化学方法）得到阐明。由于细胞中多磷酸肌醇的丰度较低，许多分析方法首先对完整细胞进行放射性标记，然后基于半个世纪前首次设计的方法，用氯仿/甲醇/水混合物提取脂质。酵母存在特殊的提取问题，因为必须打破细胞壁以促进溶剂进入并使脂质产量最大化。一旦脂质被提取出来，脂肪酸被去除，然后通过阴离子交换高效液相色谱法分析所得的水溶性甘油磷酸肌醇磷酸酯。本章描述了如何提取和定量已用[3H]肌醇放射性标记至同位素平衡的酿酒酵母细胞中的肌醇脂质。
