کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1174238 961737 2011 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
An activity-based probe for high-throughput measurements of triacylglycerol lipases
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
An activity-based probe for high-throughput measurements of triacylglycerol lipases
چکیده انگلیسی

Modulating the activity of lipases involved in the metabolism of plasma lipoproteins is an attractive approach for developing lipid raising/lowering therapies to treat cardiovascular disease. Identifying small molecule inhibitors for these membrane-active enzymes, however, is complicated by difficulties associated with measuring lipase activity and inhibition at the water–membrane interface; substrate and compound dynamics at the particle interface have the potential to confound data interpretation. Here, we describe a novel ELISA-based lipase activity assay that employs as “bait” a biotinylated active-site probe that irreversibly binds to the catalytic active-site serine of members of the triacylglycerol lipase family (hepatic lipase, lipoprotein lipase, and endothelial lipase) in solution with high affinity. Detection of “captured” (probe–enzyme) complexes on streptavidin-coated plates using labeled secondary antibodies to specific primary antibodies offers several advantages over conventional assays, including the ability to eliminate enzyme–particle and compound–particle effects; specifically measure lipase activity in complex mixtures in vitro; preferentially identify active-site-directed inhibitors; and distinguish between reversible and irreversible inhibitors through a simple assay modification. Using EL as an exemplar, we demonstrate the versatility of this assay both for high-throughput screening and for compound mechanism-of-action studies.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 414, Issue 2, 15 July 2011, Pages 254–260
نویسندگان
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