Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multi-step procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluorescence method to measure lipase activity in real time that does not require the separation of substrates from products. We developed this method using adipose triglyceride lipase (ATGL) and lipoprotein lipase (LpL) as model lipases. We first incubated a source of ATGL or LpL with substrate vesicles containing nitrobenzoxadiazole (NBD)-labeled TAG, then measured increases in NBD fluorescence and calculated enzyme activities. Incorporation of NBD-TAG into phosphatidylcholine (PC) vesicles resulted in some hydrolysis; however, incorporation of phosphatidylinositol into these NBD-TAG/PC vesicles and increasing the ratio of NBD-TAG to PC greatly enhanced substrate hydrolysis. This assay was also useful in measuring the activity of pancreatic lipase and hormone-sensitive lipase. Next, we tested several small molecule lipase inhibitors and found that orlistat inhibits all lipases, indicating that it is a pan-lipase inhibitor. In short, we describe a simple, rapid, fluorescence-based triacylglycerol hydrolysis assay to assess four major TAG hydrolases: intracellular ATGL and hormone-sensitive lipase, LpL localized at the extracellular endothelium, and pancreatic lipase present in the intestinal lumen. The major advantages of this method are its speed, simplicity, and elimination of product isolation. This assay is potentially applicable to a wide range of lipases, is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and can be used for diagnostic purposes.

中文翻译：

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一种简单、快速、灵敏的基于荧光的评估甘油三酯水解酶活性的方法。

脂肪酶是一类重要的水溶性酶，可催化疏水性三酰基甘油 (TAG) 的水解。它们的酶活性通常使用涉及分离和量化水解产物的多步骤程序来测量。我们在这里报告了一种新的荧光方法来实时测量脂肪酶活性，该方法不需要将底物与产品分离。我们使用脂肪甘油三酯脂肪酶 (ATGL) 和脂蛋白脂肪酶 (LpL) 作为模型脂肪酶开发了这种方法。我们首先将 ATGL 或 LpL 源与含有硝基苯并恶二唑 (NBD) 标记的 TAG 的底物囊泡一起孵育，然后测量 NBD 荧光的增加并计算酶活性。将 NBD-TAG 掺入到磷脂酰胆碱 (PC) 囊泡中会导致一些水解；然而，将磷脂酰肌醇掺入这些 NBD-TAG/PC 囊泡中并增加 NBD-TAG 与 PC 的比例大大增强了底物水解。该测定法还可用于测量胰脂肪酶和激素敏感性脂肪酶的活性。接下来，我们测试了几种小分子脂肪酶抑制剂，发现奥利司他对所有的脂肪酶都有抑制作用，说明它是一种泛脂肪酶抑制剂。简而言之，我们描述了一种简单、快速、基于荧光的三酰基甘油水解测定，以评估四种主要的 TAG 水解酶：细胞内 ATGL 和激素敏感脂肪酶、位于细胞外内皮的 LpL 和存在于肠腔中的胰脂肪酶。这种方法的主要优点是它的速度、简单性和消除产品隔离。该测定可能适用于广泛的脂肪酶，