A fluorescent plate reader assay for ceramide kinase

摘要

Ceramide kinase and its product ceramide 1-phosphate have been implicated in cellular proliferation and survival, activation of cytosolic phospholipase A2, mast cell degranulation, and phagocytosis. Current assays for ceramide kinase activity employ [³²P]-ATP, with separation of labelled product from excess ATP by organic extraction and thin layer chromatography. We have developed a fluorescent plate reader assay for ceramide kinase, which uses commercially available C6-NBD ceramide. Our assay is based on the differential partitioning of substrate and product following a single chloroform/methanol extraction. The product, which partitions into the aqueous phase at physiological pH, is quantitated directly in a plate reader. The substrate may be delivered using either fatty acid free albumin or detergent/lipid mixed micelles, and we have found that the use of albumin rather than detergent micelles allows one to detect lipid interactions with the enzyme that might otherwise go unnoticed. Our method is useful for assaying ceramide kinase activity both in vitro and in cultured cells, and offers several advantages over the conventional assay, including greater speed, the ability to run a larger number of assay replicates at one time, and the elimination of environmental and safety issues associated with the use of radioactive materials.