Application of phosphoinositide-binding domains for the detection and quantification of specific phosphoinositides
Application of phosphoinositide-binding domains for the detection and quantification of specific phosphoinositides
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DOI：
10.1016/j.ab.2006.05.014
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发表时间：
2006-08-01
影响因子：
2.9
通讯作者：
Takenawa, Tadaomi
中科院分区：
文献类型：
--
作者：
Furutani, Masahiro;Tsujita, Kazuya;Takenawa, Tadaomi
在哺乳动物中，已知七种磷酸肌醇作为信号分子在多种细胞过程中发挥着至关重要的作用。它们的合成和降解被认为受到磷酸肌醇激酶和磷酸酶等代谢酶的严格控制，它们的异常活性会导致疾病。因此，人们对方便且高通量地测量此类活性以筛选增强或阻断它们的药物产生了极大的兴趣。迄今为止，释放的无机磷酸盐的放射性标记和比色检测主要分别用于测量磷酸肌醇激酶和磷酸酶活性。在这里，我们描述了一种通过磷酸肌醇结合域检测和定量单个磷酸肌醇的新方法，该结合域对该脂质表现出高特异性和亲和力。酶联免疫吸附测定孔经过烷基链 (C16) 修饰，可以更均匀、定量地将含磷酸肌醇的脂质体固定到孔表面。作为底物或产物的磷酸肌醇通过特异性结合每种磷酸肌醇的pleckstrin同源结构域进行检测。通过该方法，可以比传统方法以更高的灵敏度测量磷酸肌醇含量。更重要的是，可以测量纯化酶和粗制细胞裂解物的磷酸肌醇激酶和磷酸酶活性。该测定方法简单、灵敏且定量，因此可在开发诊断测试或筛选可能由异常磷酸肌醇代谢引起的癌症和糖尿病等疾病的治疗方法方面具有多种应用。 (c) 2006 Elsevier Inc. 保留所有权利。
In mammals, seven phosphoinositides are known to play crucial roles as signaling molecules in a variety of cellular processes. Their synthesis and degradation are thought to be strictly controlled by metabolic enzymes such as phosphoinositide kinases and phosphatases, and their aberrant activities cause diseases. Thus, there is great interest in convenient and high-throughput measurement of such activities for the screening of drugs that enhance or block them. To date, radioactive labeling and colorimetric detection of released inorganic phosphates are mainly used to measure phosphoinositide kinase and phosphatase activities, respectively. Here, we describe a novel method for detecting and quantifying individual phosphoinositides via phosphoinositide-binding domains that exhibit high specificity and affinity toward this lipid. Enzyme-linked immunosorbent assay wells are modified with alkyl chains (C16), which enables more uniform and quantitative immobilization of phosphoinositide-containing liposomes onto the well surfaces. Phosphoinositides, as the substrate or the product, are detected by pleckstrin homology domains that specifically bind to each phosphoinositide. By this method, phosphoinositide contents are measured with higher sensitivities than those by conventional methods. More importantly, both phosphoinositide kinase and phosphatase activities can be measured for purified enzymes and crude cellular lysates. This assay is easy, sensitive, and quantitative and thus may have a variety of applications in the development of diagnostic tests or the screening of therapeutic treatments for diseases such as cancer and diabetes which may be caused by abnormal phosphoinositide metabolism. (c) 2006 Elsevier Inc. All rights reserved.