| Popis: |
Measurements of sphingolipid metabolism are most accurately performed by LC-MS. However, this technique is expensive, not widely accessible, and without the use of specific probes, it does not provide insight into metabolic flux through the pathway. Employing the fluorescent ceramide analogue NBD-C6-ceramide as a tracer in intact cells, we developed a comprehensive HPLC-based method that simultaneously measures the main nodes of ceramide metabolism in the Golgi. Hence, by quantifying the conversion of NBD-C6-ceramide to NBD-C6-sphingomyelin, NBD-C6-hexosylceramides, and NBD-C6-ceramide-1-phosphate (NBD-C1P), the activities of Golgi resident enzymes sphingomyelin synthase 1, glucosylceramide synthase, and ceramide kinase (CERK) could be measured simultaneously. Importantly, the detection of NBD-C1P allowed us to quantify CERK activity in cells, a usually difficult task. By applying this method, we evaluated the specificity of commonly used sphingolipid inhibitors and discovered that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, which targets glucosylceramide synthase, and fenretinide (4HPR), an inhibitor for dihydroceramide desaturase, also suppress CERK activity. This study demonstrates the benefit of an expanded analysis of ceramide metabolism in the Golgi, and it provides a qualitative and easy-to-implement method. |
