Measuring Activity of Phosphoinositide Lipid Kinases Using a Bioluminescent ADP-Detecting Assay.
Measuring Activity of Phosphoinositide Lipid Kinases Using a Bioluminescent ADP-Detecting Assay.
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使用生物发光 ADP 检测测定法测量磷酸肌醇脂质激酶的活性。
DOI：
10.1007/978-1-4939-3073-9_6
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Vidugiriene,Jolanta
中科院分区：
文献类型：
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作者：
Tai,AndrewW;Vidugiriene,Jolanta
磷脂酰肌醇（PI）及其磷酸化衍生物统称为磷酸肌苷，是参与多种细胞过程的重要第二信使，包括细胞增殖、凋亡、代谢和迁移。这些衍生物是由磷酸肌苷脂质激酶（PIKs）家族产生的。由于这些激酶在信号通路中的核心作用，测量其活性的分析通常用于药物开发。脂激酶底物存在于体内独特的膜环境中，不溶于水溶液。因此，在开发成功的脂激酶试验中最重要的考虑因素是脂激酶底物的物理状态。在这里，我们描述了两大类脂质激酶的脂质底物的制备，磷脂酰肌醇3-激酶（pi3k）和磷脂酰肌醇4-激酶（PI4Ks）。以PI4Ks为例，我们还提供了从瞬时转染的哺乳动物细胞中进行小规模激酶表达和亲和纯化的详细方案。为了测量脂激酶活性，我们采用了一种通用的生物发光ADP检测方法。该方法与多种脂质底物兼容，可作为测量所有类脂质和蛋白激酶的单一集成平台。
Phosphatidylinositol (PI) and its phosphorylated derivatives, collectively called phosphoinositides, are important second messengers involved in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Due to the central role of these kinases in signaling pathways, assays for measuring their activity are often used for drug development. Lipid kinase substrates are present in unique membrane environmentsin vivoand are insoluble in aqueous solutions. Therefore the most important consideration in developing successful lipid kinase assays is the physical state of lipid kinase substrates. Here we describe the preparation of lipid substrates for two major classes of lipid kinases, phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 4-kinases (PI4Ks). Using PI4Ks as an example, we also provide a detailed protocol for small-scale kinase expression and affinity purification from transiently transfected mammalian cells. For measuring lipid kinase activity we apply a universal bioluminescent ADP detection approach. The approach is compatible with diverse lipid substrates and can be used as a single integrated platform for measuring all classes of lipid and protein kinases.