Gramicidin pores report the activity of membrane-active enzymes.
Gramicidin pores report the activity of membrane-active enzymes.
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DOI：
10.1021/ja904072s
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发表时间：
2009-11-11
影响因子：
15
通讯作者：
Mayer, Michael
中科院分区：
文献类型：
--
作者：
Majd, Sheereen;Yusko, Erik C.;MacBriar, Alexander D.;Yang, Jerry;Mayer, Michael
磷脂酶是一类普遍存在的膜活性酶，在细胞信号传导、增殖和膜运输中起关键作用。异常磷脂酶活性与一系列疾病有关，包括癌症、炎症和心肌疾病。因此，这些酶的表征是重要的，无论是提高磷脂酶催化的理解，并加快制药和生物技术应用。本文介绍了一种新的方法来监测，在原位和实时，磷脂酶D（PLD）和磷脂酶C（PLC）的平面脂双层的活性。该方法是基于酶诱导的脂质双层电荷的变化和脂质膜附近离子浓度的伴随变化。该方法通过离子通道形成肽短杆菌肽A的孔中的离子电导的可测量变化来报告局部离子浓度的这些变化。因此，这种酶测定利用短杆菌肽孔的扩增特性来感测皮摩尔至纳摩尔浓度的膜活性酶的活性，而不需要标记底物或产物。所得的方法在脂质双层上进行，而不需要洗涤剂，在几分钟内定量天然脂质底物上的酶活性，并提供了对两个小叶的明确的脂质双层的独特访问;这种方法也使得有可能产生具有横向脂质不对称性的平面脂质双层。
Phospholipases constitute a ubiquitous class of membrane-active enzymes that play a key role in cellular signaling, proliferation, and membrane trafficking. Aberrant phospholipase activity is implicated in a range of diseases including cancer, inflammation, and myocardial disease. Characterization of these enzymes is therefore important, both for improving the understanding of phospholipase catalysis, and for accelerating pharmaceutical and biotechnological applications. This paper describes a novel approach to monitor, in-situ and in real-time, the activity of phospholipase D (PLD) and phospholipase C (PLC) on planar lipid bilayers. This method is based on enzyme-induced changes in the electrical charge of lipid bilayers and on the concomitant change in ion concentration near lipid membranes. The approach reports these changes in local ion concentration by a measurable change in the ion conductance through pores of the ion channel-forming peptide gramicidin A. This enzyme assay hence takes advantage of the amplification characteristics of gramicidin pores to sense the activity of picomolar to nanomolar concentrations of membrane-active enzymes without requiring labeling of substrates or products. The resulting method proceeds on lipid bilayers without the need for detergents, quantifies enzyme activity on native lipid substrates within minutes, and provides unique access to both leaflets of well-defined lipid bilayers; this method also makes it possible to generate planar lipid bilayers with transverse lipid asymmetry.
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