A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity
Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluoresc...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2021-01-01
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| Series: | Journal of Lipid Research |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227521000973 |
| _version_ | 1819320409955237888 |
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| author | Sujith Rajan Hazel C. de Guzman Thomas Palaia Ira J. Goldberg M. Mahmood Hussain |
| author_facet | Sujith Rajan Hazel C. de Guzman Thomas Palaia Ira J. Goldberg M. Mahmood Hussain |
| author_sort | Sujith Rajan |
| collection | DOAJ |
| description | Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep 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. |
| first_indexed | 2024-12-24T11:19:08Z |
| format | Article |
| id | doaj.art-99dd043a3c594d828f16d52f27640493 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-24T11:19:08Z |
| publishDate | 2021-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-99dd043a3c594d828f16d52f276404932022-12-21T16:58:18ZengElsevierJournal of Lipid Research0022-22752021-01-0162100115A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activitySujith Rajan0Hazel C. de Guzman1Thomas Palaia2Ira J. Goldberg3M. Mahmood Hussain4Department of Foundations of Medicine, NYU Long Island School of Medicine, and Diabetes and Obesity Research Center, NYU Langone Hospitals - Long Island, Mineola, NY, USADepartment of Foundations of Medicine, NYU Long Island School of Medicine, and Diabetes and Obesity Research Center, NYU Langone Hospitals - Long Island, Mineola, NY, USA; Department of Environmental Medicine, NYU Grossman School of Medicine, New York, NY, USADepartment of Foundations of Medicine, NYU Long Island School of Medicine, and Diabetes and Obesity Research Center, NYU Langone Hospitals - Long Island, Mineola, NY, USADivision of Endocrinology, Department of Medicine, NYU Grossman School of Medicine, New York, NY, USADepartment of Foundations of Medicine, NYU Long Island School of Medicine, and Diabetes and Obesity Research Center, NYU Langone Hospitals - Long Island, Mineola, NY, USA; VA New York Harbor Healthcare System, Brooklyn, NY, USA; For correspondence: M. Mahmood HussainLipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep 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.http://www.sciencedirect.com/science/article/pii/S0022227521000973lipidstriacylglycerolATGLenzyme activityNBD-TAGlipoprotein lipase |
| spellingShingle | Sujith Rajan Hazel C. de Guzman Thomas Palaia Ira J. Goldberg M. Mahmood Hussain A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity Journal of Lipid Research lipids triacylglycerol ATGL enzyme activity NBD-TAG lipoprotein lipase |
| title | A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity |
| title_full | A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity |
| title_fullStr | A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity |
| title_full_unstemmed | A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity |
| title_short | A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity |
| title_sort | simple rapid and sensitive fluorescence based method to assess triacylglycerol hydrolase activity |
| topic | lipids triacylglycerol ATGL enzyme activity NBD-TAG lipoprotein lipase |
| url | http://www.sciencedirect.com/science/article/pii/S0022227521000973 |
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