A comprehensive measure of Golgi sphingolipid flux using NBD C₆-ceramide: evaluation of sphingolipid inhibitors.

ABSTRACT: 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

SUBMITTER: Lee AH 

PROVIDER: S-EPMC11326893 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature